**Mutational Analysis of Effectors Encoded by Monopartite Begomoviruses and Their Satellites**

Muhammad Shafiq Shahid, Pradeep Sharma and Masato Ikegami

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/54518

## **1. Introduction**

216 Plant Science

[97] Kawalek A, Dmochowska-Boguta M, Nadolska-Orczyk A, Orczyk W. A New BSMV-Based Vector with Modified β Molecule Allows Simultaneous and Stable Silencing of

[98] Ding XS, Schneider WL, Chaluvadi SR, Mian MAR, Nelson RS. Characterization of a Brome Mosaic Virus Strain and Its Use as a Vector for Gene Silencing in Monocotyledonous Hosts. Molecular Plant Microbe Interactions 2006;19(11): 1229-39. [99] van der Linde K, Kastner C, Kumlehn J, Kahmann R, Doehlemann G. Systemic Virus-Induced Gene Silencing Allows Functional Characterization of Maize Genes During

Biotrophic Interaction with Ustilago Maydis. New Phytologist 2011;189: 471-83. [100] Lu R, Malcuit I, Moffett P, Ruiz MT, Peart J, Wu A, Rathjen JP, Bendahmane A, Day L, Baulcombe DC. High Throughput Virus-Induced Gene Silencing Implicates Heat Shock

Protein 90 in Plant Disease Resistance. The EMBO Journal 2003;22(21): 5690-9. [101] Meng Y, Moscou MJ, Wise RP. Blufensin1 Negatively Impacts Basal Defense in

[102] Yuan C, Li C, Yan L, Jackson AO, Liu Z, Han C, Yu J, Li D. A High Throughput Barley Stripe Mosaic Virus Vector for Virus Induced Gene Silencing in Monocots and Dicots.

[103] Hensel G, Himmelbach A, Chen W, Douchkov DK, Kumlehn J. Transgene Expression

[104] Joshi RL, Joshi V, Ow DW. BSMV Genome Mediated Expression of a Foreign Gene in

[105] Choi I, Stenger DC, Morris TJ, French R. A Plant Virus Vector for Systemic Expression

[106] Lawrence DM, Jackson AO. Interactions of the TGB1 Protein during Cell-to-Cell Movement of Barley Stripe Mosaic Virus. Journal of Virology 2001;75(18): 8712-23. [107] Haupt S, Duncan GM, Holzberg S, Oparka KJ. Sieve-Element Unloading in Sink

[108] Lawrence DM, Jackson AO. Requirements for Cell-To-Cell Movement of Barley Stripe Mosaic Virus in Monocot and Dicot Hosts. Molecular Plant Pathology 2001;(2): 65-75. [109] Manning VA, Chu AL, Scofield SR, Ciuffetti LM. Intracellular Expression of a Host-Selective Toxin, ToxA, in Diverse Plants Phenocopies Silencing of a ToxA-Interacting

[110] Tai Y, Bragg J. Dual Applications of a Virus Vector for Studies of Wheat-Fungal

[111] Donald RGK, Jackson AO. The Barley Stripe Mosaic Virus CB Gene Encodes a Multifunctional Cysteine-Rich Protein that Affects Pathogenesis. Plant Cell 1994;6: 1593-

[112] Ryan MD, King AM, Thomas GP. Cleavage of Foot-and-Mouth Disease Virus Polyprotein is Mediated by Residues Located within a 19 Amino Acid Sequence.

[113] Donnelly ML, Hughes LE, Luke G, Mendoza H, ten Dam E, Gani D, Ryan MD. The 'Cleavage' Activities of Foot-and-Mouth Disease Virus 2A Site-Directed Mutants and Naturally Occurring '2A-Like' Sequences. Journal of General Virology 2001;82(Pt 5):

Systems in the Triticeae Cereals. Journal of Plant Physiology 2011;168: 30-44.

Dicot and Monocot Plant Cells. The EMBO Journal 1990;9(9): 2663-9.

of Foreign Genes in Cereals. The Plant Journal 2000;23(4): 547-55.

Leaves of Barley is Symplastic. Plant Physiology 2001;125: 209-18.

Protein, ToxABP1. New Phytologist 2010;187: 1034-47.

Journal of General Virology 1991;72(Pt 11): 2727-32.

Interactions. Biotechnology 2007;6(2): 288-91.

606.

1027-41.

Response to Barley Powdery Mildew. Plant Physiology 2009;149: 271-85.

PLoS ONE 2011; 6(10): 1-16.

Two Genes. Cellular & Molecular Biology Letters 2012;17(1): 107-23.

The geminiviruses are plant-infecting viruses with genomes consisting of circular, singlestranded DNA (ssDNA) geminate particles [86]. Members of the family *Geminiviridae* have been grouped into four genera (*Begomovirus, Curtovirus, Mastrevirus and Topocuvirus*) based on genome organization, host range and insect vector [29, 87]. The majority of geminiviruses belong to the genus Begomovirus, are transmitted by whiteflies (*Bemisia tabaci*: Gennadius), and infect dicotyledonous plant species [85]. The monopartite begomovirus genome is ∼2.8 kb nucleotides in length and encode genes both in complementary and virion sense from a non-coding intergenic region that contains promoter sequences and the origin (*ori*) of virionstrand DNA replication. The *ori* consists of a predicted hairpin structure that contains the absolutely conserved (for geminiviruses) nonanucleotide (TAATATTAC) loop sequence and repeated motifs upstream known as iterons.

## **2. Functions of effectors encoded by monopartite begomoviruses**

### **2.1. Complementary-sense**

The complementary-sense strand encodes the Rep proteins, also known as C1, AC1 and AL1, is a multifunctional protein and the only viral protein absolutely required for virus replication. Rep is encoded on the complementary sense strand (Fig. 1 DNA A). This protein is involved in several biological processes: initiation and termination of rolling circle replication (RCR) by nicking and religating the replication origin of viral DNA [45] and repression of its own gene transcription [19]. The Rep proteins of geminiviruses are closely related and show substantial sequence conservation. Four functional domains have been delineated for begomovirus Rep : the N-terminal domain (amino acids 1 to 120), which is involved in initiation by geminiviruses [63], AC1 protein initiates rolling circle replication

by a site-specific cleavage within the loop of the conserved nonamer sequence, TAATATTAC [30]. The AC1 protein binding site is located between the TATA box and the transcription start site for the *Rep* gene and acts as the origin recognition sequence and as a negatively regulatory element for *AC1* gene transcription [19], the oligomerization domain (121 to 180 aa), leading to interactions with itself and with host factors [28]. The AC1 protein alone can initiate RCR without requiring other accessory viral factors [34]. AC1 protein also has DNA helicase activity which depends upon the oligomeric state of the protein [14].

Mutational Analysis of Effectors Encoded by Monopartite Begomoviruses and Their Satellites 219

In contrast to New World (NW) begomoviruses, Old World (OW) begomoviruses have AV2/V2 and this is involved in the movement of monopartite viruses. A recent report shown that the V2 (a homolog of AV2) of a monopartite begomovirus is involved in overcoming host defenses mediated by post-transcriptional gene silencing as well as in movement [114, 115]. V2 targets a step in the RNA silencing pathway which is subsequent to

Recently, the majority of the begomoviruses originating from the OW have been shown to be monopartite and to associate with a class of ssDNA satellites known as betasatellites (earlier known as DNAβ) [9]. Betasatellites are approximately half the size of their helper begomoviruses (approx.1.4 kb) and are required by the helper virus to induce typical disease symptoms in their original hosts. The success of begomovirus-betasatellite disease complexes appears to be due to the promiscuous nature of betasatellites that allows them to be *trans*-replicated by several distinct begomoviruses [53, 59]. These begomovirusbetasatellite disease complexes are widespread throughout OW and outnumber bipartite begomoviruses whereas in the NW only bipartite begomoviruses are native. There have been recent reports which showed that betasatellite can complement the function of DNA B, suggesting that the betasatellite may provide movement functions to its helper begomovirus [74]. Betasatellite can be associated with distinct begomoviruses and it can interact and

*Tomato leaf curl virus* (ToLCV), originating from Australia, was shown to be associated with a single-stranded DNA satellite molecule [18]. The ToLCV satellite (ToLCV-sat) is approximately 682 nt in length and sequence unrelated to ToLCV and it depends on ToLCV for replication and encapsidation. It has no discernable effects on viral replication or symptoms caused by ToLCV. Betasatellites have three structural features: a approx.115 bp highly conserved region, *βC1* gene and a region rich in adenine, [76, 108] (Fig. 1 betasatellite). This gene has the capacity to encode a 13-14-kDa protein comprising 118 amino acids, although some betasatellites have additional N-terminal amino acids [79, 108]. Recently it has been shown that betasatellite to be pathogenicity determinant and suppressor of RNA silencing [16, 66]. It also induced abnormal cell division in *N. benthamiana* [17]. Betasatellites do not contain the iterons of their helper begomoviruses, although betasatellite clearly must possess sequences that are recognized by the

begomovirus-encoded Rep in order to allow transreplication of the betasatellite [76].

All the reported betasatellites [54] or defective betasatellites (half size of wild type betasatellite) [7] contain the A-rich region, the A-rich region may play biological role in betasatellites [95]. A-rich region is not required for *trans*-replication of betasatellite and not related with encapsidation also. However, the A-rich region deleted mutant caused milder symptom [95]. The begomovirus accumulates to normal levels in Ageratum in the presence

make new complex with diverse monopartite begomoviruses [110-112].

the Dicer-mediated cleavage of dsRNA [109, 70].

**3. Role of effectors encoded by satellites** 

**3.1. Betasatellite** 

The transcriptional activator protein (TrAP); is also known as AC2, C2 an AL2. AC2 is a 15- KD a transcriptional activator protein unique to begomoviruses because it is absent in mastreviruses and a related protein in curtoviruses, AC2 protein, seems to play a different role. In mastreviruses, AC1 protein provide the functions of AC2 [51]. TrAP is necessary for transactivation of late genes [90]. Recently, several researchers have shown that the AC2 gene of *Cabbage leaf curl virus* (CaLCuV) activates the CP promoter in mesophyll and acts to derepress the promoter in vascular tissue, similar to that observed for TGMV [44]. Further, since AC2 1-100 is as effective a suppressor as the full-length AC2 protein, activation and silencing suppression appear to be independent activities. For example Gopal et al. [26] showed that AC2 of *Bhindi yellow vein mosaic virus* (BYVMV) is involved in transactivation and only mildly in suppression of gene silencing of monopartite begomoviruses viruses and not in transmission.

The replication enhancer protein (REn); also named as AC3/AL3. AC3 is a 16 KD a protein in curtoviruses and in begomoviruses. The AC3 protein greatly enhances viral DNA accumulation of curtoviruses and begomoviruses [22, 92] by interacting with Rep [81]. Experimental observations suggested that AC3 protein might increase the affinity of Rep for the origin. Complementation studies revealed that AC3 could act on heterologous viruses [93].

The C4 protein, for which the function remains unclear but for some viruses is a pathogenicity determinant and a suppressor of PTGS [73]. AC4 is highly variable among begomoviruses, which is expressed from an open reading frame (ORF) embedded in the Rep ORF.

#### **2.2. Virion-sense**

The virion-sense strand encodes the genes required for insect transmission and movement in plants, coat protein (CP) and V2 protein. Monopartite begomovirus capsids are composed of a single CP, encoded by the *V1* gene or (also known as AV1), depending on the geminivirus [107]. For monopartite geminiviruses, CP is essential for systemic spread through the plant [12]. Besides the encapsidation function, CP is also required for transmission of the virus between the plants. The CP of the monopartite begomoviruses facilitates the transfer of infecting viral DNA into the host cell nucleus and is essential for systemic virus movement [5, 46, 50, 104]. The CP also determines the vector specificity [10, 32, 33] and protects the viral ssDNA from degradation during transmission by the insect vector [3], or mechanical inoculation [24].

In contrast to New World (NW) begomoviruses, Old World (OW) begomoviruses have AV2/V2 and this is involved in the movement of monopartite viruses. A recent report shown that the V2 (a homolog of AV2) of a monopartite begomovirus is involved in overcoming host defenses mediated by post-transcriptional gene silencing as well as in movement [114, 115]. V2 targets a step in the RNA silencing pathway which is subsequent to the Dicer-mediated cleavage of dsRNA [109, 70].

## **3. Role of effectors encoded by satellites**

#### **3.1. Betasatellite**

218 Plant Science

not in transmission.

[93].

ORF.

**2.2. Virion-sense** 

vector [3], or mechanical inoculation [24].

by a site-specific cleavage within the loop of the conserved nonamer sequence, TAATATTAC [30]. The AC1 protein binding site is located between the TATA box and the transcription start site for the *Rep* gene and acts as the origin recognition sequence and as a negatively regulatory element for *AC1* gene transcription [19], the oligomerization domain (121 to 180 aa), leading to interactions with itself and with host factors [28]. The AC1 protein alone can initiate RCR without requiring other accessory viral factors [34]. AC1 protein also has DNA helicase activity which depends upon the oligomeric state of the protein [14].

The transcriptional activator protein (TrAP); is also known as AC2, C2 an AL2. AC2 is a 15- KD a transcriptional activator protein unique to begomoviruses because it is absent in mastreviruses and a related protein in curtoviruses, AC2 protein, seems to play a different role. In mastreviruses, AC1 protein provide the functions of AC2 [51]. TrAP is necessary for transactivation of late genes [90]. Recently, several researchers have shown that the AC2 gene of *Cabbage leaf curl virus* (CaLCuV) activates the CP promoter in mesophyll and acts to derepress the promoter in vascular tissue, similar to that observed for TGMV [44]. Further, since AC2 1-100 is as effective a suppressor as the full-length AC2 protein, activation and silencing suppression appear to be independent activities. For example Gopal et al. [26] showed that AC2 of *Bhindi yellow vein mosaic virus* (BYVMV) is involved in transactivation and only mildly in suppression of gene silencing of monopartite begomoviruses viruses and

The replication enhancer protein (REn); also named as AC3/AL3. AC3 is a 16 KD a protein in curtoviruses and in begomoviruses. The AC3 protein greatly enhances viral DNA accumulation of curtoviruses and begomoviruses [22, 92] by interacting with Rep [81]. Experimental observations suggested that AC3 protein might increase the affinity of Rep for the origin. Complementation studies revealed that AC3 could act on heterologous viruses

The C4 protein, for which the function remains unclear but for some viruses is a pathogenicity determinant and a suppressor of PTGS [73]. AC4 is highly variable among begomoviruses, which is expressed from an open reading frame (ORF) embedded in the Rep

The virion-sense strand encodes the genes required for insect transmission and movement in plants, coat protein (CP) and V2 protein. Monopartite begomovirus capsids are composed of a single CP, encoded by the *V1* gene or (also known as AV1), depending on the geminivirus [107]. For monopartite geminiviruses, CP is essential for systemic spread through the plant [12]. Besides the encapsidation function, CP is also required for transmission of the virus between the plants. The CP of the monopartite begomoviruses facilitates the transfer of infecting viral DNA into the host cell nucleus and is essential for systemic virus movement [5, 46, 50, 104]. The CP also determines the vector specificity [10, 32, 33] and protects the viral ssDNA from degradation during transmission by the insect Recently, the majority of the begomoviruses originating from the OW have been shown to be monopartite and to associate with a class of ssDNA satellites known as betasatellites (earlier known as DNAβ) [9]. Betasatellites are approximately half the size of their helper begomoviruses (approx.1.4 kb) and are required by the helper virus to induce typical disease symptoms in their original hosts. The success of begomovirus-betasatellite disease complexes appears to be due to the promiscuous nature of betasatellites that allows them to be *trans*-replicated by several distinct begomoviruses [53, 59]. These begomovirusbetasatellite disease complexes are widespread throughout OW and outnumber bipartite begomoviruses whereas in the NW only bipartite begomoviruses are native. There have been recent reports which showed that betasatellite can complement the function of DNA B, suggesting that the betasatellite may provide movement functions to its helper begomovirus [74]. Betasatellite can be associated with distinct begomoviruses and it can interact and make new complex with diverse monopartite begomoviruses [110-112].

*Tomato leaf curl virus* (ToLCV), originating from Australia, was shown to be associated with a single-stranded DNA satellite molecule [18]. The ToLCV satellite (ToLCV-sat) is approximately 682 nt in length and sequence unrelated to ToLCV and it depends on ToLCV for replication and encapsidation. It has no discernable effects on viral replication or symptoms caused by ToLCV. Betasatellites have three structural features: a approx.115 bp highly conserved region, *βC1* gene and a region rich in adenine, [76, 108] (Fig. 1 betasatellite). This gene has the capacity to encode a 13-14-kDa protein comprising 118 amino acids, although some betasatellites have additional N-terminal amino acids [79, 108]. Recently it has been shown that betasatellite to be pathogenicity determinant and suppressor of RNA silencing [16, 66]. It also induced abnormal cell division in *N. benthamiana* [17]. Betasatellites do not contain the iterons of their helper begomoviruses, although betasatellite clearly must possess sequences that are recognized by the begomovirus-encoded Rep in order to allow transreplication of the betasatellite [76].

All the reported betasatellites [54] or defective betasatellites (half size of wild type betasatellite) [7] contain the A-rich region, the A-rich region may play biological role in betasatellites [95]. A-rich region is not required for *trans*-replication of betasatellite and not related with encapsidation also. However, the A-rich region deleted mutant caused milder symptom [95]. The begomovirus accumulates to normal levels in Ageratum in the presence of betasatellite suggesting that the satellite functions either by facilitating the replication or movement of the begomovirus or by suppressing a host defense mechanism such as gene silencing. Recently it has been shown that a betasatellite can override the AC4 pathogenicity phenotype of TLCV and it can complement the function of DNA B [73]. Despite its importance to the disease phenotype, there is still no information available concerning even the most fundamental properties of the satellite.

Mutational Analysis of Effectors Encoded by Monopartite Begomoviruses and Their Satellites 221

Many begomovirus betasatellite complexes are also associated with a third ssDNA component for which the collective term alphasatellite (earlier known as DNA 1; R.W. Briddon, manuscript in preparation). However, alphasatellites are dispensable for virus infection and appear to play no significant role in the etiology of the diseases with which they are associated [56]. Alphasatellite components are satellite-like, circular ssDNA molecules approx.1375 nucleotides in length (Fig.1 alphasatellite). They encode a single gene, a rolling circle replication initiator protein (Rep), and are capable of autonomous replication in plant cells. Closely related to the replication associated protein encoding components of nanoviruses (a second family of plant infecting ssDNA viruses), from which they are believed to have evolved, they require a helper begomovirus for movement within

Several alphasatellites are capable of replicating and systemically infecting their plant host in the presence of a helper begomovirus without a visible effect on symptom development or virulence [6, 40]. However AYVSGA a different type of 'DNA-2'-class alphasatellite that ameliorates symptom severity in an infected host and also capable of reducing virulence and the relative accumulation of its associated Tomato leaf curl betasatellite (ToLCB) [1]. Alphasatellites have been acquired by helper begomoviruses to restrain virulence to achieve

Recently, two 'DNA-1-type' alphasatellites Gossypium mustelinium symptomless alphasatellite (GMusSLA) and Gossypium darwinii symptomless alphasatellite (GDarSLA) phylogenetically divergent from the DNA-2-type alphasatellite have each been shown to attenuate symptoms caused by their helper begomovirus [60]. However [35] hypothesize that symptom attenuation and a relative reduction in betasatellite accumulation might result from DNA-2-mediated modulation of betasatellite activity. Possibly alphasatellite modulates begomovirus-betasatellite pathogenicity by interfering with βC1, a key virulence factor [8]. Also alphasatellite rep can interact with C4 of CLCuRaV that might be providing an additional possible mechanism for symptom amelioration by alphasatellites. Furthermore alpha-Rep down regulate betasatellite replication (In the field), and thus downregulation of the manifestation of the pathogenicity determinant βC1 [60], moreover alpha-Rep proteins GMusSLA and GDarSLA can act as a strong suppression of posttranscriptional

Post-transcriptional gene silencing (PTGS) which is initiated by double stranded RNA (dsRNA) is common in plant–virus interactions and is an evolutionarily conserved mechanism that protects host cells against invasive nucleic acids, such as viruses, transposons and transgenes [100]. As a counter to this host defense, most plant viruses encode proteins which act as suppressors of PTGS [71]. Viral suppressors of PTGS interfere with various steps of this pathway including initiation, maintenance or systemic silencing

**4. Alphasatellites** 

and between plants [56, 80].

increased viral fitness [76, 105].

gene silencing (PTGS) [60].

**5. Post-transcriptional gene silencing (PTGS)** 

which are mainly downstream of dsRNA production [52, 57].

**Figure 1.** Genome organization of monopartite begomoviruses-satellite complex. DNA-A (encoding replication-associated protein [Rep], coat protein [CP], replication enhancer protein [REn], transcriptional activator protein [TrAP] and proteins possibly involved in virus movement [AV2], pathogenicity determinant and a suppressor of RNA silencing [AC4], viral genome replication [AC5]) Alphasatellites are self-replicating molecules encoding their own Rep. Betasatellites are dependent on their helper viruses for their replication and encode a single protein, βC1, which upregulate replication of helper virus and suppress host defense. Both satellites have an A-rich region and in addition to this betasatellites have a region of sequence conserved between all examples known as the satellite conserved region (SCR).

In Arabidopsis, these pathways are affected by the DICER- like proteins (DCL1, DCL2, and DCL3) that are nuclear localized and are required for miRNA and siRNA biogenesis. Thus, C1 protein may affect the activity of the DICER-like proteins in plants during nuclear activities that function in silencing suppression. The other possibilities are that C1 protein could down-regulate transcription of a host protein that acts in the PTGS pathway in the cytoplasm or that C1 protein could activate transcription of a host PTGS inhibitor [15].

## **4. Alphasatellites**

220 Plant Science

conserved region (SCR).

the most fundamental properties of the satellite.

of betasatellite suggesting that the satellite functions either by facilitating the replication or movement of the begomovirus or by suppressing a host defense mechanism such as gene silencing. Recently it has been shown that a betasatellite can override the AC4 pathogenicity phenotype of TLCV and it can complement the function of DNA B [73]. Despite its importance to the disease phenotype, there is still no information available concerning even

**Figure 1.** Genome organization of monopartite begomoviruses-satellite complex. DNA-A (encoding

In Arabidopsis, these pathways are affected by the DICER- like proteins (DCL1, DCL2, and DCL3) that are nuclear localized and are required for miRNA and siRNA biogenesis. Thus, C1 protein may affect the activity of the DICER-like proteins in plants during nuclear activities that function in silencing suppression. The other possibilities are that C1 protein could down-regulate transcription of a host protein that acts in the PTGS pathway in the cytoplasm or that C1 protein could activate transcription of a host PTGS inhibitor [15].

replication-associated protein [Rep], coat protein [CP], replication enhancer protein [REn], transcriptional activator protein [TrAP] and proteins possibly involved in virus movement [AV2], pathogenicity determinant and a suppressor of RNA silencing [AC4], viral genome replication [AC5]) Alphasatellites are self-replicating molecules encoding their own Rep. Betasatellites are dependent on their helper viruses for their replication and encode a single protein, βC1, which upregulate replication of helper virus and suppress host defense. Both satellites have an A-rich region and in addition to this betasatellites have a region of sequence conserved between all examples known as the satellite

Many begomovirus betasatellite complexes are also associated with a third ssDNA component for which the collective term alphasatellite (earlier known as DNA 1; R.W. Briddon, manuscript in preparation). However, alphasatellites are dispensable for virus infection and appear to play no significant role in the etiology of the diseases with which they are associated [56]. Alphasatellite components are satellite-like, circular ssDNA molecules approx.1375 nucleotides in length (Fig.1 alphasatellite). They encode a single gene, a rolling circle replication initiator protein (Rep), and are capable of autonomous replication in plant cells. Closely related to the replication associated protein encoding components of nanoviruses (a second family of plant infecting ssDNA viruses), from which they are believed to have evolved, they require a helper begomovirus for movement within and between plants [56, 80].

Several alphasatellites are capable of replicating and systemically infecting their plant host in the presence of a helper begomovirus without a visible effect on symptom development or virulence [6, 40]. However AYVSGA a different type of 'DNA-2'-class alphasatellite that ameliorates symptom severity in an infected host and also capable of reducing virulence and the relative accumulation of its associated Tomato leaf curl betasatellite (ToLCB) [1]. Alphasatellites have been acquired by helper begomoviruses to restrain virulence to achieve increased viral fitness [76, 105].

Recently, two 'DNA-1-type' alphasatellites Gossypium mustelinium symptomless alphasatellite (GMusSLA) and Gossypium darwinii symptomless alphasatellite (GDarSLA) phylogenetically divergent from the DNA-2-type alphasatellite have each been shown to attenuate symptoms caused by their helper begomovirus [60]. However [35] hypothesize that symptom attenuation and a relative reduction in betasatellite accumulation might result from DNA-2-mediated modulation of betasatellite activity. Possibly alphasatellite modulates begomovirus-betasatellite pathogenicity by interfering with βC1, a key virulence factor [8]. Also alphasatellite rep can interact with C4 of CLCuRaV that might be providing an additional possible mechanism for symptom amelioration by alphasatellites. Furthermore alpha-Rep down regulate betasatellite replication (In the field), and thus downregulation of the manifestation of the pathogenicity determinant βC1 [60], moreover alpha-Rep proteins GMusSLA and GDarSLA can act as a strong suppression of posttranscriptional gene silencing (PTGS) [60].

## **5. Post-transcriptional gene silencing (PTGS)**

Post-transcriptional gene silencing (PTGS) which is initiated by double stranded RNA (dsRNA) is common in plant–virus interactions and is an evolutionarily conserved mechanism that protects host cells against invasive nucleic acids, such as viruses, transposons and transgenes [100]. As a counter to this host defense, most plant viruses encode proteins which act as suppressors of PTGS [71]. Viral suppressors of PTGS interfere with various steps of this pathway including initiation, maintenance or systemic silencing which are mainly downstream of dsRNA production [52, 57].

RNA silencing in plants operates as an antiviral defense response; to establish infection, viruses must suppress RNA silencing by the host [100]. Begomoviruses have been shown to induce PTGS in infected plants by producing virus specific siRNAs (21, 22 and 24 nt) [97]. To counteract this host defence, geminiviruses encode RNA silencing suppressors [4]. However, depending on each intrinsic virus and its interaction with plant host factors, the efficacy of virus-induced PTGS may vary [99]. At least three RNA-silencing suppressors have been reported in TYLCD-associated or related begomoviruses. Thus, the V2 protein of TYLCV functions as an RNA-silencing suppressor; it counteracts the innate immune response of the host plant by interacting with SISGS3, the tomato homolog of the Arabidopsis SGS3 protein involved in the RNA-silencing pathway. The TrAP protein of the related monopartite begomovirus. *Tomato yellow leaf curl China virus* (TYLCCNV) is also involved in suppression of RNA silencing [98], probably by activating transcription of host genes that control silencing [97]. The C4 protein of the monopartite begomoviruses ToLCV, *Ageratum yellow vein virus* (AYVV), and *Bhendi yellow vein mosaic virus* (BiYVMV) also have the ability to suppress RNA silencing [26, 84].

Mutational Analysis of Effectors Encoded by Monopartite Begomoviruses and Their Satellites 223

**Mutation Position Type of**

**mutation** 

frameshift

frameshift

deletion

inversion

revertion

**References** 

frameshift Rigden et al., 1993

deletion Rigden et al., 1993

1994

1996

al.,1997

1998

1998

2001

al., 2004

**Virus Accession**

TYLCV-

TYLCV-

TYLCV-

TYLCV-

AYVV-

[SG:pHN419:97]

Sic[IT:pSic36:95]

Sar[ES:Psp95:93]

Sar[ES:Psp95:93]

Sar[ES:Psp95:93]

**number** 

TLCV-[AU] AF084006 V1 N-terminal/

TLCV-[AU] AF084006 C4 N-terminal

**Mutated ORF** 

V2 N-terminal/

*Bam*HI

*Bgl*II

*Bam*HI

(at 2457&2463)

Z25751 C4 C>T at 2432 point Jupin et al.,

Z25751 C2 ∆CC2>31 deletion Noris et al.,

Z28390 CP H134 substitution Noris et al.,

Z25751 CP Q134H site-directedNoris et al.,

X74516 C4 A>T at 2419 C4mut substitution Saunders et

2457&2463)

 C2 ∆NC2>33 deletion C2 ∆C2>33-104 deletion

 V1 156+GATC frameshift V1 324C>T stop V2 748>CTAG stop

RepC1 L198 substitution

 CP ∆CCP>150 deletion V1 ∆NV1>63 deletion V1 ∆CV1>84 deletion C4 ∆NC4>54 deletion C4 ∆CC4>60 deletion

 V/V2 N-terminal/*Bgl*II/ *Bam*HI

V/V2 C-terminal/*Bgl*II/

 V/V2 C-terminal/*Bam*HI/ *Bgl*II

C4 N-terminal (at

C4 C>T at 2423

TYLCV-Sar[FR:98] X61153 C2 1523+GATC frameshift Wartig et

TYLCV-DO[DO:99] AF024715 CP ∆NCP>180 deletion Rojas et al.,

## **6. Mutagenesis of effectors encoded by monopartite begomoviruses**

Little is known about gene function in monopartite begomoviruses. However, gene function has been studied extensively in other types of geminiviruses which share organization and nucleotide sequence similarities with TYLCV. Mutational analysis of few monopartite begomoviruses like TYLCV define similarities and differences between this single component geminivirus and bipartite geminiviruses in functions essential for systemic spread and infectivity [103]. The CP appeared to be required for systemic movement of TYLCV in *N. benthamiana* and tomato, consistent with those of mutation analyses obtained with other monopartite geminiviruses such as MSV, BCTV, and TLCV [5, 12, 69, 61] have shown that *Tomato yellow leaf curl virus*-Sardinia (TYLCV-Sar) C2 can form stable complexes with ssDNA (and less preferably with dsDNA) and that the binding is sequence nonspecific. AC2 of TYLCV have also been involved in the activation of other viral genes and was considered as a transcriptional activator [91]. However, transcription factors usually show high sequence specificity.

TYLCV ORF V1 truncated either 133 nt upstream or 19 nt downstream of the initiation codon of ORF V2 would altered the viral DNA forms, it suggested that the V1 protein may participate in the switch from dsDNA to ssDNA synthesis. Indeed, interaction between V1 and the CP has already been proposed, in view of the concerted evolution of these two protein sequences following a geo-graphical gradient of similarity [39], and the synergistic reduction in ssDNA levels of a TYLCV V1-V2 double mutant compared to single mutants [69]. Although TYLCV V1 mutants did not greatly overproduce dsDNA, the similarity of phenotype between BCTV V2 and TYLCV V1 mutants may indicated that the two corresponding gene products serve a related function. It has shown [69] (Table 1) has shown that disruption of the V1 gene in the monopartite Australian isolate of TLCV did not affect its ability to spread in tomato, although the infection was asymptomatic and the DNA levels reduced.

the ability to suppress RNA silencing [26, 84].

high sequence specificity.

reduced.

RNA silencing in plants operates as an antiviral defense response; to establish infection, viruses must suppress RNA silencing by the host [100]. Begomoviruses have been shown to induce PTGS in infected plants by producing virus specific siRNAs (21, 22 and 24 nt) [97]. To counteract this host defence, geminiviruses encode RNA silencing suppressors [4]. However, depending on each intrinsic virus and its interaction with plant host factors, the efficacy of virus-induced PTGS may vary [99]. At least three RNA-silencing suppressors have been reported in TYLCD-associated or related begomoviruses. Thus, the V2 protein of TYLCV functions as an RNA-silencing suppressor; it counteracts the innate immune response of the host plant by interacting with SISGS3, the tomato homolog of the Arabidopsis SGS3 protein involved in the RNA-silencing pathway. The TrAP protein of the related monopartite begomovirus. *Tomato yellow leaf curl China virus* (TYLCCNV) is also involved in suppression of RNA silencing [98], probably by activating transcription of host genes that control silencing [97]. The C4 protein of the monopartite begomoviruses ToLCV, *Ageratum yellow vein virus* (AYVV), and *Bhendi yellow vein mosaic virus* (BiYVMV) also have

**6. Mutagenesis of effectors encoded by monopartite begomoviruses** 

Little is known about gene function in monopartite begomoviruses. However, gene function has been studied extensively in other types of geminiviruses which share organization and nucleotide sequence similarities with TYLCV. Mutational analysis of few monopartite begomoviruses like TYLCV define similarities and differences between this single component geminivirus and bipartite geminiviruses in functions essential for systemic spread and infectivity [103]. The CP appeared to be required for systemic movement of TYLCV in *N. benthamiana* and tomato, consistent with those of mutation analyses obtained with other monopartite geminiviruses such as MSV, BCTV, and TLCV [5, 12, 69, 61] have shown that *Tomato yellow leaf curl virus*-Sardinia (TYLCV-Sar) C2 can form stable complexes with ssDNA (and less preferably with dsDNA) and that the binding is sequence nonspecific. AC2 of TYLCV have also been involved in the activation of other viral genes and was considered as a transcriptional activator [91]. However, transcription factors usually show

TYLCV ORF V1 truncated either 133 nt upstream or 19 nt downstream of the initiation codon of ORF V2 would altered the viral DNA forms, it suggested that the V1 protein may participate in the switch from dsDNA to ssDNA synthesis. Indeed, interaction between V1 and the CP has already been proposed, in view of the concerted evolution of these two protein sequences following a geo-graphical gradient of similarity [39], and the synergistic reduction in ssDNA levels of a TYLCV V1-V2 double mutant compared to single mutants [69]. Although TYLCV V1 mutants did not greatly overproduce dsDNA, the similarity of phenotype between BCTV V2 and TYLCV V1 mutants may indicated that the two corresponding gene products serve a related function. It has shown [69] (Table 1) has shown that disruption of the V1 gene in the monopartite Australian isolate of TLCV did not affect its ability to spread in tomato, although the infection was asymptomatic and the DNA levels



Mutational Analysis of Effectors Encoded by Monopartite Begomoviruses and Their Satellites 225

replacement in the RepC1 protein. The other mutation located at nt 708 (C instead of G), determining a CAG-to-CAC codon change in the CP mRNA and a Q134H replacement in the CP. This indicated that the Q134H mutation changed a viral DNA, only capable of replicating in single cells (Sic), into one that was systemically infectious, but not insect transmissible (SicRcv). Comparative analysis of Sic, SicRcv, and the hybrid genomes and showed that the mutation in the CP gene, not in the Rep gene, was responsible for restoring infectivity in SicRcv; however, it still did not result in a whitefly-transmissible TYLCV. In TYLCV-Sar, the two capsid protein alterations resulted in the same either non-infectious or non-transmissible phenotype. Mutants containing the combinations QQ, QH, and PH at positions 129 and 134 were infectious in plants, whereas those with PQ are not. The PQ mutants can replicate and accumulate CP and V2 protein in leaf discs, but appear unable to produce virus particles. Mutants having the PH combination at positions 129 and 134 infect plants and form apparently normal virions, but are not transmissible by whiteflies. Changing the amino acid at position 152 (D or E) does not influence the phenotype. Requirement of the CP for infection has been demonstrated previously [62] suggested that accurate particle assembly is also necessary. In fact, the PQ mutants, which are unable to assemble virions, accumulate CP in leaf discs, showing that its expression and stability were not altered. Another TYLCV protein, V2, for which a role in virus assembly has recently

For example Rojas et al. [70] has shown that C4, V1, and CP gene may function in TYLCV-DO movement. The CP localized to nuclei and nucleoli and was found to act as a nuclear shuttle, mediating the import and export of DNA [70]. It was consistent with results obtained for the TYLCV CP in heterologous experimental systems [43, 68]. Recently, Liu et al [49] also showed the same behavior for the CP of the monopartite mastrevirus, MSV. TYLCV CP was found to accumulate in the nucleolus and the absence of the N-and Cterminal CP mutants from the nucleolus implicates CP motifs in this localization. As the nucleolus is the site of rRNA synthesis and packaging of ribosomal proteins, it may also serve as the site of geminiviral replication/gene expression [70]. The TYLCV C4 targeted to the cell periphery and/or cell wall, consistent with a role in cell-to-cell movement of viral

Disruption of the AYVV C4 ORF (A>T at position 2419nt) alters the phenotype in agroinoculated *N. benthamiana* from upward leaf roll and vein swelling to downward leaf curl [79] (Table 2). Previously,[88] also found the identical functions of BCTV C4 ORF mutant in this host. The AYVV C4 proteins may perform partially redundant functions involving convergent pathways and the behavior of ACMV AC4 and TYLCCNV C4 is

For example Stanley and Latham [58] have shown that V2 protein of *Papaya leaf curl virus* (PaLCuV) is potentially involved in the elicitation of cell death response. The deletion mutants (having deletions of 32 and 60 amino acids, respectively, at the N-terminal end of V2) exhibited a systemic HR in *Nicotiana benthamiana* plants. While C-terminal end deletions of 60 , 80 and 119 amino acids abolished the induction of HR, however 50 amino acids deletion

been, suggested [103].

DNA [65, 75, 101].

consistent with such a function [98].

**Table 1.** List of published studies reporting deletion mutants of monopartite begomoviruses

For example Noris et al. [62] suggested that the region of the CP between amino acids 129 and 134 is essential for both the correct assembly of virions and transmission by the insect vector. The genome of the SicRcv (infectious) had the same size as the original Sic DNA 9 (non-infectious) differed by only 2 nt. One change was at nt 2025 (A instead of T in the plus strand), determining a CAC-to-CUC codon change in the RepC1 mRNA and an H198L replacement in the RepC1 protein. The other mutation located at nt 708 (C instead of G), determining a CAG-to-CAC codon change in the CP mRNA and a Q134H replacement in the CP. This indicated that the Q134H mutation changed a viral DNA, only capable of replicating in single cells (Sic), into one that was systemically infectious, but not insect transmissible (SicRcv). Comparative analysis of Sic, SicRcv, and the hybrid genomes and showed that the mutation in the CP gene, not in the Rep gene, was responsible for restoring infectivity in SicRcv; however, it still did not result in a whitefly-transmissible TYLCV. In TYLCV-Sar, the two capsid protein alterations resulted in the same either non-infectious or non-transmissible phenotype. Mutants containing the combinations QQ, QH, and PH at positions 129 and 134 were infectious in plants, whereas those with PQ are not. The PQ mutants can replicate and accumulate CP and V2 protein in leaf discs, but appear unable to produce virus particles. Mutants having the PH combination at positions 129 and 134 infect plants and form apparently normal virions, but are not transmissible by whiteflies. Changing the amino acid at position 152 (D or E) does not influence the phenotype. Requirement of the CP for infection has been demonstrated previously [62] suggested that accurate particle assembly is also necessary. In fact, the PQ mutants, which are unable to assemble virions, accumulate CP in leaf discs, showing that its expression and stability were not altered. Another TYLCV protein, V2, for which a role in virus assembly has recently been, suggested [103].

224 Plant Science

TYLCV-Mld[ES:72:97]

**Virus Accession**

**number** 

**Mutated ORF** 

V2 C-terminal

PaLCuV-[PK:02] AJ436992 V2 N-terminal (1-32) deletion Mubin et al.,

TYLCV-IL[IL:89] X15656 CP Lys-Thr CPmut3 substitution Yaakov et al.,

ToLCJV-A[ID] AB100304 CP CPΔ191-257 deletion Sharma et al.,

ToLCJV-A[ID] AB100304 V2 N-terminal (58aa) Sharma et al.,

For example Noris et al. [62] suggested that the region of the CP between amino acids 129 and 134 is essential for both the correct assembly of virions and transmission by the insect vector. The genome of the SicRcv (infectious) had the same size as the original Sic DNA 9 (non-infectious) differed by only 2 nt. One change was at nt 2025 (A instead of T in the plus strand), determining a CAC-to-CUC codon change in the RepC1 mRNA and an H198L

 V2 C-terminal (58aa) ToLCJV-A[ID] AB100304 V2 pGEMV2ΔC Sharma et al.,

**Table 1.** List of published studies reporting deletion mutants of monopartite begomoviruses

 CP CPΔ1-30/Δ50-257 deletion CP CPΔ1-30/Δ191-257 deletion

V2 N-terminal (1-60) deletion

 V2 C-terminal (111-149) deletion V2 C-terminal (101-149) deletion V2 C-terminal (91-149) deletion V2 C-terminal (71-149) deletion V2 C-terminal (30-149) deletion

 CP Arg-Pro CPmut4 substitution CP Arg-Leu CPmut19 substitution

 CP CPΔ1-190 deletion CP CPΔ31-257 deletion

 CP CPΔ1-31 deletion CP CPΔ16-20 deletion CP CPΔ1-190/Δ245-250 deletion CP CPΔ1-30/Δ62-257 deletion

AF071228 C4 C>G at 9 C4mut substitution Tomas et al.,

(130-149)

**Mutation Position Type of**

**mutation** 

deletion

**References** 

2010

2011

2011

2009

2010

2011

For example Rojas et al. [70] has shown that C4, V1, and CP gene may function in TYLCV-DO movement. The CP localized to nuclei and nucleoli and was found to act as a nuclear shuttle, mediating the import and export of DNA [70]. It was consistent with results obtained for the TYLCV CP in heterologous experimental systems [43, 68]. Recently, Liu et al [49] also showed the same behavior for the CP of the monopartite mastrevirus, MSV. TYLCV CP was found to accumulate in the nucleolus and the absence of the N-and Cterminal CP mutants from the nucleolus implicates CP motifs in this localization. As the nucleolus is the site of rRNA synthesis and packaging of ribosomal proteins, it may also serve as the site of geminiviral replication/gene expression [70]. The TYLCV C4 targeted to the cell periphery and/or cell wall, consistent with a role in cell-to-cell movement of viral DNA [65, 75, 101].

Disruption of the AYVV C4 ORF (A>T at position 2419nt) alters the phenotype in agroinoculated *N. benthamiana* from upward leaf roll and vein swelling to downward leaf curl [79] (Table 2). Previously,[88] also found the identical functions of BCTV C4 ORF mutant in this host. The AYVV C4 proteins may perform partially redundant functions involving convergent pathways and the behavior of ACMV AC4 and TYLCCNV C4 is consistent with such a function [98].

For example Stanley and Latham [58] have shown that V2 protein of *Papaya leaf curl virus* (PaLCuV) is potentially involved in the elicitation of cell death response. The deletion mutants (having deletions of 32 and 60 amino acids, respectively, at the N-terminal end of V2) exhibited a systemic HR in *Nicotiana benthamiana* plants. While C-terminal end deletions of 60 , 80 and 119 amino acids abolished the induction of HR, however 50 amino acids deletion induced local necrosis, but not systemic. The mutants with 20 and 40 amino acids deletion produce HR both at the inoculated and in newly emerged leaves, although the systemic symptoms for the 40-amino-acid deletion mutant were delayed and were milder. The amino acid sequences between positions 92 and 101 are essential for the elicitation of HR, whereas those between 102 and 115 affect the timing and severity of the response. V2 of PaLCuV at amino acid positions 116 and 118 contains a conserved CxC. Mutations of this motif have been shown to abolish both the pathogenicity and suppressor of RNA silencing activities of the protein [64, 109]. Phosphorylation of MPs may also play a role in controlling the switch from viral replication to translation [36, 37]. Few earlier studies showed that for PaLCuV V2, deletion of sequences encompassing this motif abrogates the ability to induce HR [58].

Mutational Analysis of Effectors Encoded by Monopartite Begomoviruses and Their Satellites 227

U74627 ∆nt 35-146 (112nt) deletation Li et al., 2007

deletation

deletation

deletation

deletation

site-derected

site-derected

site-derected

Saunders et al., 2004

site-derected Cui et al., 2004

**Satellite Accession region (nt.) Type of References number mutation** 

548 βC1mut1

G>T at 486 βC1mut2

(CIM-F)

(ACIM-S)

(CIM-B)

(151nt)

(197nt)

(105nt)

AYVJB-[ID:04] AB162142 4>ATGtga stop Kont et al., 2007 AYVB-[SG:pBS-beta:99] AJ252072 794-795 deletation Saunders et al., 2008 118-119 deletation 804-806 deletation 801-1047 deletation 1048-1051 deletation 1146-1147 deletation 1146-1150 deletation 1269-1271 deletation 1229-1234 deletation TbCSVB-[CN:Y35:01] AJ420318 DNA∆C1β Qian et al., 2008

∆nt 296-420 (1251nt)

∆nt 35-296 (262nt) deletation

∆nt 492-540 (49nt) deletation

GAA>TAG (CIM-T)

ATG (2) >ATC (2)

TYLCCNB-[CN:Y10:01] AJ421621 742-952 deletation Xiaorong et al., 2004 TYLCCNB-[CN:Y10:01] AJ421621 ∆C1β truncation Qian and Zhou 2005 CLCuVβ-[PK:00] AJ298903 195-484 site-derected Saeed et al., 2005 504-596 stop 586 frame-shift ATG>ATA stop BYVMB-[IN:Muth:01] AJ308425 51-140 ∆NβC1 deletation Kumar et al., 2006 1-80 ∆CβC1 deletation

ACT>TGA

AYVB-[SG:pBS-beta:99] AJ252072 AT>TA at 547/

TYLCCNB-[CN:Y10:01] AJ421621 ATG>ATC

∆nt 146-296

∆nt 296-492

∆nt 540-641

TYLCV-satDNA-

[AU:96]

The first 30 N-terminal amino acids of the TYLCV-IL CP are needed for nuclear import of the protein into the plant cell, suggesting the CP's involvement in nuclear shuttling of the virus genome [43]. This was confirmed by the finding of a strong interaction between the CP and the plant nuclear import receptor karyopherin α1 (Kap α1) [94]. The TYLCV CP has been found to inter act with itself (CP–CP or homotypic interaction) which may be important for capsule assembly as it is made up solely of CP units serving as building blocks. Mutations in the TYLCV-IL V1 gene coding for the TYLCV-IL CP by replacing Lys with Thr, Arg with Pro, and Arg with Leu, according to the positions of amino acids mutated [31]. TYLCV CP mutated failed to interact with the w.t. CP, while the w.t. protein showed strong homo typic interaction. As the CP has been suggested to be a shuttle protein for the viral genome into the plant cell nucleus [43, 70], its interaction with the nucleartransport mediator Kap α1 is an important step and has been shown to occur at high affinity [94]. A mutation in the NLS domain, in particular at Arg19, disrupts the CP's interaction with proteins that are known to interact with the w.t. CP [106.]. Earlier Sharma et al. [113-115] demonstrated by the constructed a series of single and double deletions into the coding sequence of *Tomato leaf curl Java virus* ToLCJAV-A[ID] CP and found that, amino acids (aa)16KVRRR20 in the N-terminal region of CP functioned as nuclear/nucleolar localization signals (NLSs). Further, the region from aa 52RKPR55 contained basic amino acid cluster was capable to redirect the CP to the nucleus. Deletion mutant analysis revealed that this property was attributed to a nuclear export signal (NES) sequence consisted of aa (245LKIRIY250) reside at C-terminal part of CP. Additionally ToLCJV V2 is a target of host defense responses. Deletion of 58 amino acids (aa) from the N-terminus did not affect the HR, suggesting that this region has no role in the HR, while deletion of 58 aa from the Cterminus of V2 abolished both the HR response and V2 silencing suppressor activity, suggesting that these sequences are required for the HR-like response and suppression of PTGS. He also demonstrated that ToLCJV V2 is a pathogenicity determinant that elicits an HR-like response. Further deletion analysis that fusion of Nterminal part of the V2, containing the nuclear export signals (NES), directed the accumulation of fluorescence towards the cell cytoplasm. Also V2 enhances the coat protein-mediated nuclear export of ToLCJV and is consistent with the model in which V2 mediates viral DNA export from the nucleus to the plasmodesmata.

Mutational Analysis of Effectors Encoded by Monopartite Begomoviruses and Their Satellites 227

226 Plant Science

HR [58].

nucleus to the plasmodesmata.

induced local necrosis, but not systemic. The mutants with 20 and 40 amino acids deletion produce HR both at the inoculated and in newly emerged leaves, although the systemic symptoms for the 40-amino-acid deletion mutant were delayed and were milder. The amino acid sequences between positions 92 and 101 are essential for the elicitation of HR, whereas those between 102 and 115 affect the timing and severity of the response. V2 of PaLCuV at amino acid positions 116 and 118 contains a conserved CxC. Mutations of this motif have been shown to abolish both the pathogenicity and suppressor of RNA silencing activities of the protein [64, 109]. Phosphorylation of MPs may also play a role in controlling the switch from viral replication to translation [36, 37]. Few earlier studies showed that for PaLCuV V2, deletion of sequences encompassing this motif abrogates the ability to induce

The first 30 N-terminal amino acids of the TYLCV-IL CP are needed for nuclear import of the protein into the plant cell, suggesting the CP's involvement in nuclear shuttling of the virus genome [43]. This was confirmed by the finding of a strong interaction between the CP and the plant nuclear import receptor karyopherin α1 (Kap α1) [94]. The TYLCV CP has been found to inter act with itself (CP–CP or homotypic interaction) which may be important for capsule assembly as it is made up solely of CP units serving as building blocks. Mutations in the TYLCV-IL V1 gene coding for the TYLCV-IL CP by replacing Lys with Thr, Arg with Pro, and Arg with Leu, according to the positions of amino acids mutated [31]. TYLCV CP mutated failed to interact with the w.t. CP, while the w.t. protein showed strong homo typic interaction. As the CP has been suggested to be a shuttle protein for the viral genome into the plant cell nucleus [43, 70], its interaction with the nucleartransport mediator Kap α1 is an important step and has been shown to occur at high affinity [94]. A mutation in the NLS domain, in particular at Arg19, disrupts the CP's interaction with proteins that are known to interact with the w.t. CP [106.]. Earlier Sharma et al. [113-115] demonstrated by the constructed a series of single and double deletions into the coding sequence of *Tomato leaf curl Java virus* ToLCJAV-A[ID] CP and found that, amino acids (aa)16KVRRR20 in the N-terminal region of CP functioned as nuclear/nucleolar localization signals (NLSs). Further, the region from aa 52RKPR55 contained basic amino acid cluster was capable to redirect the CP to the nucleus. Deletion mutant analysis revealed that this property was attributed to a nuclear export signal (NES) sequence consisted of aa (245LKIRIY250) reside at C-terminal part of CP. Additionally ToLCJV V2 is a target of host defense responses. Deletion of 58 amino acids (aa) from the N-terminus did not affect the HR, suggesting that this region has no role in the HR, while deletion of 58 aa from the Cterminus of V2 abolished both the HR response and V2 silencing suppressor activity, suggesting that these sequences are required for the HR-like response and suppression of PTGS. He also demonstrated that ToLCJV V2 is a pathogenicity determinant that elicits an HR-like response. Further deletion analysis that fusion of Nterminal part of the V2, containing the nuclear export signals (NES), directed the accumulation of fluorescence towards the cell cytoplasm. Also V2 enhances the coat protein-mediated nuclear export of ToLCJV and is consistent with the model in which V2 mediates viral DNA export from the



Mutational Analysis of Effectors Encoded by Monopartite Begomoviruses and Their Satellites 229

the nonanucleotide sequence) by Rep to initiate rolling-circle replication of the virion strand. Similarly, deletion of betasatellite sequences from 1130 to 116 that is conserved (between all betasatellites) stopped the betasatellite's ability to be trans-replicated and maintained by helper viruses both from OW (CLCuRaV) and New world and *Cabbage leaf curl virus* (CbLCuV). Trans-replication of CLCuMuB remained unaffected by deletion of the sequence

ToLCJAV alone can cause infection and displayed leaf curl symptoms. But, symptom expression of ToLCJAV in the presence of ToLCJAB is enhanced. In contrast, ToLCJAV and AYVB (mutated βC1) restored mild symptoms. It suggested that the βC1 protein was required for symptom induction and is a determinant of pathogenicity, βC1 protein

For example Li et al. [47] have shown the deletion mutant of TYLCV sat-DNA (from 296- 641nt) lacked the ability to replicate or replicated poorly by deleting of (region nt 35-296). Also sequence from nt 296-35 is to be essential for sat-DNA replication. The deletion of a 112 nt region downstream of the stem-loop from nt 35-146 and 151nt from 146-296 cannot effect on the replication of sat-DNA but reduced significantly. However, the deletion from nt 35- 296 regions diminished sat-DNA replication these deletions loss of genomic sequences required for replication or due to changes in genome size. Heterologous non-viral DNA fragments can restore the wild-type 682 nt sat-DNA size and of replication when the replacement occurred in the region between nt 35 and 296. However, the sequence replacements in the region nt 35 to 296 of the sat-DNA improved the accumulation of sat-DNA considerably relative to the deleted constructs in this region. The sequence elements distributed within the entire sat-DNA molecule contribute to replication activity, but that sequence elements within the region from nt 35 to 296 are dispensable for replication.

For example Saeed et al. [72] used mutagenesis study of CLCuMB and tobacco was used as the host plant rather that cotton, the natural host of CLCuB. Few studies showed that it was symptomless when inoculated with *Cotton leaf cur Multan virus* (CLCuMV) alone but showed drastic symptoms when coinoculated with CLCuMB [9]. *Nicotiana benthamiana* showed a severe symptom on inoculated with CLCuMV with or without CLCuMB. Evidence for the involvement of the βC1 ORF in modulation of symptom expression also provided by [108] demonstrated few DNA β species associated with tomato and tobacco infecting begomoviruses and found that in-frame mutation of the βC1 initiation codon resulted in loss

In recent studies Saunders et al. [79] have proved that disruption of the βC1 ORF prevented infection of the AYVB complex in ageratum and altered their phenotype in *N. benthamiana* to that produced by AYVV alone. For example Kumar et al. [42] tested the infectivity of two βC1 mutant constructs, first carrying a stop codon at amino acid position 41 and second with two stop codons at positions 9 and 41, and both resulted in loss of pathogenicity in tobacco plants on coinoculated with TLCV as helper virus. These mutation studies indicated that the βC1 ORF is involved in pathogenicity and that the expression of its N terminal 40

between coordinates 995 and 1095 by CLCuRaV [59].

of symptom severity in *N. benthamiana*.

amino acids is not sufficient for its function.

expression in *N. benthamiana* plants and as a suppressor of PTGS [41].

**Table 2.** List of published studies reporting deletion mutants of DNA Satellites

### **7. Mutational analysis of effectors encoded by satellites**

Betasatellite molecules have been associated with numerous monopartite begomoviruses in China, including *Tobacco curly shoot virus* (TbCSV) and TYLCCNV that infect tomato and tobacco field plants [108, 47]. TbCSB is not essential for infection but increases symptoms in some hosts [17, 47]. However in case of TYLCCNB which is essential for symptom induction, The βC1 gene of TYLCCNB is required for symptom induction but not for the replication of betasatellite. Also a mutated βC1 deleted is stably maintained in few hosts by TYLCCNV [17, 67]. TbCSB with the complete βC1 deleted (∆βC1) returns to a size comparable to that of the intact betasatellite in few systemically infected *N. glutinosa*, *N. tabacum Samsun* and *P. bybrida* plants plants. The levels of accumulation of the size revertant betasatellite were similar to those of ∆βC1 in same hosts (*N. benthamiana* and *N. glutinosa*) plants showing size reversion of the betasatellite developed viral symptoms similar to those induced by TbCSV and DNA∆βC1 [67]. A βC1 gene frame-shift mutant of TYLCCNVB was unable to induce disease symptoms and consequently, did not play a role in silencing suppression [67]. The complete coding region of Y10βC1 (TYLCCNB), followed by N- and C-terminal deletion mutants showed multimerization mediated by amino acids between positions 60 and 100 [13]. Karyopherin-α, a transport receptor involved in nuclear import were reported to interact with the C-terminal sequences of BYVMB- βC1 [42]. A myristoylation-like motif (GMDVNE) positioned at the C-terminal of CLCuMB-βC1 (103 to 108aa) interacted with a ubiquitin-conjugating enzyme involved in targeting proteins for degradation by the 26S proteasome [20, 78] identified sequences on AYVB by deletion mutagenesis required for trans-replication by AYVV. βC1 of Cotton leaf curl Multan betasatellite (CLCuMuB) has been shown to have possible virus movement function [74]. Generally, sequences between the *βC1* gene and the A-rich region are not essential for transreplication by begomoviruses. Nevertheless, deletion of these sequences abolish the ability of the betasatellite to upregulate virus levels in plants and the symptoms expression [59]. For geminiviruses hairpin structure that contains the nonanucleotide sequence is an essential part of the virion sense origin of replication that is recognized and nicked (within the nonanucleotide sequence) by Rep to initiate rolling-circle replication of the virion strand. Similarly, deletion of betasatellite sequences from 1130 to 116 that is conserved (between all betasatellites) stopped the betasatellite's ability to be trans-replicated and maintained by helper viruses both from OW (CLCuRaV) and New world and *Cabbage leaf curl virus* (CbLCuV). Trans-replication of CLCuMuB remained unaffected by deletion of the sequence between coordinates 995 and 1095 by CLCuRaV [59].

228 Plant Science

**Satellite Accession region (nt.) Type of References number mutation** 

 ∆1130-116 deletation ∆995-1095 deletation

βC1-∆CTD deletation

**Table 2.** List of published studies reporting deletion mutants of DNA Satellites

**7. Mutational analysis of effectors encoded by satellites** 

CLCuMA-[PK:2:99] AJ132345 915-1117 deletation Shahid et al., 2009 CLCuMuB-[PK:09] FJ607041 ∆150-840 deletation Nawaz-ul-Rehman et al.,

CLCuMB-[PK:00] AJ298903 ∆C1β deletation Kharazmi et al., 2012 TYLCCNB-[CN:Y10:01] AJ421621 N-terminal (NTG) deletation Cheng et al., 2011

C-terminal (CTG) deletation

Betasatellite molecules have been associated with numerous monopartite begomoviruses in China, including *Tobacco curly shoot virus* (TbCSV) and TYLCCNV that infect tomato and tobacco field plants [108, 47]. TbCSB is not essential for infection but increases symptoms in some hosts [17, 47]. However in case of TYLCCNB which is essential for symptom induction, The βC1 gene of TYLCCNB is required for symptom induction but not for the replication of betasatellite. Also a mutated βC1 deleted is stably maintained in few hosts by TYLCCNV [17, 67]. TbCSB with the complete βC1 deleted (∆βC1) returns to a size comparable to that of the intact betasatellite in few systemically infected *N. glutinosa*, *N. tabacum Samsun* and *P. bybrida* plants plants. The levels of accumulation of the size revertant betasatellite were similar to those of ∆βC1 in same hosts (*N. benthamiana* and *N. glutinosa*) plants showing size reversion of the betasatellite developed viral symptoms similar to those induced by TbCSV and DNA∆βC1 [67]. A βC1 gene frame-shift mutant of TYLCCNVB was unable to induce disease symptoms and consequently, did not play a role in silencing suppression [67]. The complete coding region of Y10βC1 (TYLCCNB), followed by N- and C-terminal deletion mutants showed multimerization mediated by amino acids between positions 60 and 100 [13]. Karyopherin-α, a transport receptor involved in nuclear import were reported to interact with the C-terminal sequences of BYVMB- βC1 [42]. A myristoylation-like motif (GMDVNE) positioned at the C-terminal of CLCuMB-βC1 (103 to 108aa) interacted with a ubiquitin-conjugating enzyme involved in targeting proteins for degradation by the 26S proteasome [20, 78] identified sequences on AYVB by deletion mutagenesis required for trans-replication by AYVV. βC1 of Cotton leaf curl Multan betasatellite (CLCuMuB) has been shown to have possible virus movement function [74]. Generally, sequences between the *βC1* gene and the A-rich region are not essential for transreplication by begomoviruses. Nevertheless, deletion of these sequences abolish the ability of the betasatellite to upregulate virus levels in plants and the symptoms expression [59]. For geminiviruses hairpin structure that contains the nonanucleotide sequence is an essential part of the virion sense origin of replication that is recognized and nicked (within

2009

ToLCJAV alone can cause infection and displayed leaf curl symptoms. But, symptom expression of ToLCJAV in the presence of ToLCJAB is enhanced. In contrast, ToLCJAV and AYVB (mutated βC1) restored mild symptoms. It suggested that the βC1 protein was required for symptom induction and is a determinant of pathogenicity, βC1 protein expression in *N. benthamiana* plants and as a suppressor of PTGS [41].

For example Li et al. [47] have shown the deletion mutant of TYLCV sat-DNA (from 296- 641nt) lacked the ability to replicate or replicated poorly by deleting of (region nt 35-296). Also sequence from nt 296-35 is to be essential for sat-DNA replication. The deletion of a 112 nt region downstream of the stem-loop from nt 35-146 and 151nt from 146-296 cannot effect on the replication of sat-DNA but reduced significantly. However, the deletion from nt 35- 296 regions diminished sat-DNA replication these deletions loss of genomic sequences required for replication or due to changes in genome size. Heterologous non-viral DNA fragments can restore the wild-type 682 nt sat-DNA size and of replication when the replacement occurred in the region between nt 35 and 296. However, the sequence replacements in the region nt 35 to 296 of the sat-DNA improved the accumulation of sat-DNA considerably relative to the deleted constructs in this region. The sequence elements distributed within the entire sat-DNA molecule contribute to replication activity, but that sequence elements within the region from nt 35 to 296 are dispensable for replication.

For example Saeed et al. [72] used mutagenesis study of CLCuMB and tobacco was used as the host plant rather that cotton, the natural host of CLCuB. Few studies showed that it was symptomless when inoculated with *Cotton leaf cur Multan virus* (CLCuMV) alone but showed drastic symptoms when coinoculated with CLCuMB [9]. *Nicotiana benthamiana* showed a severe symptom on inoculated with CLCuMV with or without CLCuMB. Evidence for the involvement of the βC1 ORF in modulation of symptom expression also provided by [108] demonstrated few DNA β species associated with tomato and tobacco infecting begomoviruses and found that in-frame mutation of the βC1 initiation codon resulted in loss of symptom severity in *N. benthamiana*.

In recent studies Saunders et al. [79] have proved that disruption of the βC1 ORF prevented infection of the AYVB complex in ageratum and altered their phenotype in *N. benthamiana* to that produced by AYVV alone. For example Kumar et al. [42] tested the infectivity of two βC1 mutant constructs, first carrying a stop codon at amino acid position 41 and second with two stop codons at positions 9 and 41, and both resulted in loss of pathogenicity in tobacco plants on coinoculated with TLCV as helper virus. These mutation studies indicated that the βC1 ORF is involved in pathogenicity and that the expression of its N terminal 40 amino acids is not sufficient for its function.

Disruption of the βC1 ORF of AYVB by introducing an internal in-frame nonsense codon (G>T) did not prevent transreplication and systemic movement of the βC1 mutant by AYVV in lab host (*N.benthamiana*). The mutated βC1 removed the influence of the satellite on symptom development in this host and prevented symptomatic infection of ageratum. That suggested the βC1 protein is an important pathogenicity factor that plays an essential role in the proliferation of the AYVV-betasatellite complex in its real host. For example Saunders et al. [79] also shown that βC1 ORF initiation codon (AT) to a nonsense codon (TA) did not completely eliminate betasatellite activity. A similar mutation in the βC1 ORF of a satellite associated with TYLCCNB was shown previously [108]. The βC1 ORF encodes a pathogenicity determinant that suppressed a host defense mechanism [76].

Mutational Analysis of Effectors Encoded by Monopartite Begomoviruses and Their Satellites 231

interacted with a ubiquitin-conjugating enzyme involved in targeting proteins for degradation by the 26S proteasome [21]. It also seems to indicate interference with a functionality associated with the C terminus of Y10βC1. βC1 protein of AYVB, CLCuMB or BYVMB with GFP fused at the N-terminus also presented as granular spots in the cytoplasm

TYLCCNB presumably has one or more *cis*-acting elements needed for replication and binds to TYLCCNV replication protein (Rep) for replication, and these elements are most probably located in the 115-nucleotide highly conserved region of betasatellite upstream of its stemloop structure. Recently, Astroga [2] showed that a 5-bp core sequence (GGN1N2N3) is a typical constituent of Rep-binding iterons. Conserved GG motifs occur upstream of the115 nucleotide highly conserved region of betasatellite. One or more of these GG motifs, combined with the 115 nucleotide highly conserved region, possibley responsible for Rep binding to betasatellite. However, the Rep binding activity of the TLCV-sat from Australia seems much less specific: TLCV-sat contains an A-rich region but lacks a βC1 gene and is believed to be a defective betasatellite molecule [48]. The effect of mutation of the conserved βC1 gene of

TYLCCNVB indicated that the βC1 protein plays a key role in symptom induction.

The position and size of the βC1 gene of the betasatellite molecules are conserved in all betasatellite molecules, and the mutation of the start codon of C1 gene in TYLCCNB showed that it's a pathogenicity determinant [108, 6]. Few studies has been also shown that the βC1 protein of betasatellite associated with TYLCCNV or AYVV is an essential pathogenicity determinant [17, 79], it may act as suppressors of post-transcriptional gene silencing that interfering the host defense system, thus, the presence of C1 protein facilitates efficient infection of the virus in hosts [102]. For example Tau and Zhou [96] showed that *βC1* gene was not required for the TYLCCNV and betasatellite replication and truncated betasatellite molecules with the deletion of the entire *βC1* gene were stable in infected plants. Defective DNAs, betasatellite and alphasatellite associated with begomoviruses are maintained at approximately half the size of the genomic components [83, 89, 55, 80, 77]. Some proofs have been displayed that geminiviruses CP can encapsidate circular ssDNA molecules of about half or quarter the size of the genomic DNA [18, 51]. Immunocapture PCR indicated that the truncated TYLCCNB of about 1 kb in length may be encapsidated with TYLCCNV coat

βC1of BYVMB have a nuclear export or peripheral localization function and βC1 interacts with itself, also with CP and the tomato protein karyopherin α. Mutagenesis of βC1 protein showed that the domain of βC1 interacting with CP is at the N terminal half whereas the domain(s) of βC1interacting with itself and karyopherin α are at the C terminal half and the role of BYVMD βC1 as a suppressor of posttranscriptional gene silencing was explored [42]. Karyopherins are soluble transport receptors that interact with basic NLS sequences and help in nuclear import [27]. Full length betasatellite of CLCuMB can substitute for the movement function of the DNA B of a bipartite begomovirus *Tomato leaf cur New Dehli virus DNA-B* (ToLCNDV DNA-B). However, the betasatellite containing a disrupted βC1 ORF did not mobilize the DNA A for systemic infection, suggested that the βC1 protein was required

and around the nucleus [42, 84].

protein in vivo [67].

for movement [74].

For example [78] have demonstrated that the region of AYVB between the introduced nt 114 and 1047 sites is not required for betasatellite replication. This region includes the βC1 open reading frame (ORF), which encodes a gene essential for pathogenicity [79] and an A-rich region that may serve to maintain the size integrity of the satellite [76]. For example [78] found that the entire ORF is dispensable and is consistent with the findings of [67] for the betasatellite associated with TYLCCNV. In addition, removal of the A-rich region from TYLCCNB was tolerated, although the deletion mutant was associated with milder infection than those produced by the wild-type satellite [95]. In contrast, deletion of this region in AYVB did not affect the phenotype, at least in *N. benthamiana*. Maximum deletions within non coding regions of the begomovirus genome were not tolerated and the deletion mutants revert to wild type size by both intra-and intermolecular recombination during systemic movement [23, 25,]. For example Saunders et al. [78] also demonstrated removal of 361 nt of betasatellite representing 27% of the satellite and the region between nt 1047 and 1146 is important for betasatellite replication. It contained an inverted repeat flanking a sequence that is identical to the ToLCV iteron ToLCV sat-DNA [18]. Protein binding assays followed by mutagenesis have demonstrated that this motif in both ToLCV and sat-DNA represents a high affinity Rep binding site, although it is not required for replication of either the begomovirus or its satellite [48]. Saunders and associates [80] found that the region between nt 1146-1229 and sequences across the nt 1268 of AYVB are also required for replication.

SCR is highly conserved nature between distinct satellites [typically above 65% sequence identity with blocks of absolutely conserved sequence [7] strongly suggests that it also plays an important role in the virus replication cycle. In addition, the adjacent stem-loop and conserved nonanucleotide sequence would be expected to participate in replication. Approximately the 386 nt upstream of the stem–loop structure in ToLCV sat-DNA, as well as the stem–loop structure itself, are essential for replication [47].

βC1 is a multi-functional protein encoded by betasatellites that are associated with the majority of monopartite begomoviruses [11]. For example Cheng et al. [13] proved by deletion mutants of Y10βC1 that multimerization was mediated by amino acids between positions 60 and 100. Previous studies say that the C-terminal sequences of BYVMB-βC1 were interact with karyopherin α, a transport receptor involved in nuclear import [42]. A myristoylation-like motif (GMDVNE) located at the C-terminal of CLCuMB-βC1 (103-108aa) interacted with a ubiquitin-conjugating enzyme involved in targeting proteins for degradation by the 26S proteasome [21]. It also seems to indicate interference with a functionality associated with the C terminus of Y10βC1. βC1 protein of AYVB, CLCuMB or BYVMB with GFP fused at the N-terminus also presented as granular spots in the cytoplasm and around the nucleus [42, 84].

230 Plant Science

Disruption of the βC1 ORF of AYVB by introducing an internal in-frame nonsense codon (G>T) did not prevent transreplication and systemic movement of the βC1 mutant by AYVV in lab host (*N.benthamiana*). The mutated βC1 removed the influence of the satellite on symptom development in this host and prevented symptomatic infection of ageratum. That suggested the βC1 protein is an important pathogenicity factor that plays an essential role in the proliferation of the AYVV-betasatellite complex in its real host. For example Saunders et al. [79] also shown that βC1 ORF initiation codon (AT) to a nonsense codon (TA) did not completely eliminate betasatellite activity. A similar mutation in the βC1 ORF of a satellite associated with TYLCCNB was shown previously [108]. The βC1 ORF encodes a

For example [78] have demonstrated that the region of AYVB between the introduced nt 114 and 1047 sites is not required for betasatellite replication. This region includes the βC1 open reading frame (ORF), which encodes a gene essential for pathogenicity [79] and an A-rich region that may serve to maintain the size integrity of the satellite [76]. For example [78] found that the entire ORF is dispensable and is consistent with the findings of [67] for the betasatellite associated with TYLCCNV. In addition, removal of the A-rich region from TYLCCNB was tolerated, although the deletion mutant was associated with milder infection than those produced by the wild-type satellite [95]. In contrast, deletion of this region in AYVB did not affect the phenotype, at least in *N. benthamiana*. Maximum deletions within non coding regions of the begomovirus genome were not tolerated and the deletion mutants revert to wild type size by both intra-and intermolecular recombination during systemic movement [23, 25,]. For example Saunders et al. [78] also demonstrated removal of 361 nt of betasatellite representing 27% of the satellite and the region between nt 1047 and 1146 is important for betasatellite replication. It contained an inverted repeat flanking a sequence that is identical to the ToLCV iteron ToLCV sat-DNA [18]. Protein binding assays followed by mutagenesis have demonstrated that this motif in both ToLCV and sat-DNA represents a high affinity Rep binding site, although it is not required for replication of either the begomovirus or its satellite [48]. Saunders and associates [80] found that the region between nt 1146-1229

pathogenicity determinant that suppressed a host defense mechanism [76].

and sequences across the nt 1268 of AYVB are also required for replication.

as the stem–loop structure itself, are essential for replication [47].

SCR is highly conserved nature between distinct satellites [typically above 65% sequence identity with blocks of absolutely conserved sequence [7] strongly suggests that it also plays an important role in the virus replication cycle. In addition, the adjacent stem-loop and conserved nonanucleotide sequence would be expected to participate in replication. Approximately the 386 nt upstream of the stem–loop structure in ToLCV sat-DNA, as well

βC1 is a multi-functional protein encoded by betasatellites that are associated with the majority of monopartite begomoviruses [11]. For example Cheng et al. [13] proved by deletion mutants of Y10βC1 that multimerization was mediated by amino acids between positions 60 and 100. Previous studies say that the C-terminal sequences of BYVMB-βC1 were interact with karyopherin α, a transport receptor involved in nuclear import [42]. A myristoylation-like motif (GMDVNE) located at the C-terminal of CLCuMB-βC1 (103-108aa) TYLCCNB presumably has one or more *cis*-acting elements needed for replication and binds to TYLCCNV replication protein (Rep) for replication, and these elements are most probably located in the 115-nucleotide highly conserved region of betasatellite upstream of its stemloop structure. Recently, Astroga [2] showed that a 5-bp core sequence (GGN1N2N3) is a typical constituent of Rep-binding iterons. Conserved GG motifs occur upstream of the115 nucleotide highly conserved region of betasatellite. One or more of these GG motifs, combined with the 115 nucleotide highly conserved region, possibley responsible for Rep binding to betasatellite. However, the Rep binding activity of the TLCV-sat from Australia seems much less specific: TLCV-sat contains an A-rich region but lacks a βC1 gene and is believed to be a defective betasatellite molecule [48]. The effect of mutation of the conserved βC1 gene of TYLCCNVB indicated that the βC1 protein plays a key role in symptom induction.

The position and size of the βC1 gene of the betasatellite molecules are conserved in all betasatellite molecules, and the mutation of the start codon of C1 gene in TYLCCNB showed that it's a pathogenicity determinant [108, 6]. Few studies has been also shown that the βC1 protein of betasatellite associated with TYLCCNV or AYVV is an essential pathogenicity determinant [17, 79], it may act as suppressors of post-transcriptional gene silencing that interfering the host defense system, thus, the presence of C1 protein facilitates efficient infection of the virus in hosts [102]. For example Tau and Zhou [96] showed that *βC1* gene was not required for the TYLCCNV and betasatellite replication and truncated betasatellite molecules with the deletion of the entire *βC1* gene were stable in infected plants. Defective DNAs, betasatellite and alphasatellite associated with begomoviruses are maintained at approximately half the size of the genomic components [83, 89, 55, 80, 77]. Some proofs have been displayed that geminiviruses CP can encapsidate circular ssDNA molecules of about half or quarter the size of the genomic DNA [18, 51]. Immunocapture PCR indicated that the truncated TYLCCNB of about 1 kb in length may be encapsidated with TYLCCNV coat protein in vivo [67].

βC1of BYVMB have a nuclear export or peripheral localization function and βC1 interacts with itself, also with CP and the tomato protein karyopherin α. Mutagenesis of βC1 protein showed that the domain of βC1 interacting with CP is at the N terminal half whereas the domain(s) of βC1interacting with itself and karyopherin α are at the C terminal half and the role of BYVMD βC1 as a suppressor of posttranscriptional gene silencing was explored [42]. Karyopherins are soluble transport receptors that interact with basic NLS sequences and help in nuclear import [27]. Full length betasatellite of CLCuMB can substitute for the movement function of the DNA B of a bipartite begomovirus *Tomato leaf cur New Dehli virus DNA-B* (ToLCNDV DNA-B). However, the betasatellite containing a disrupted βC1 ORF did not mobilize the DNA A for systemic infection, suggested that the βC1 protein was required for movement [74].

## **8. Potential of mutated satellites using a virus induced gene silencing vectors**

Mutational Analysis of Effectors Encoded by Monopartite Begomoviruses and Their Satellites 233

also expression) of inserted sequences [82]. Rolling-circle replication initiator protein of

The monopartite begomovirus associated with DNA-satellites (Betasatellite and Alphasatellite ) complex is in the norm throughout the Old World, particularly in South Asian countries. The epidemiology and evolution of this complex has been extensively analyzed since its first description. Monopartite begomovirus encoded all the genes needed to cause a successful infection. Many of these genes are coding for multifunctional proteins, adding another level of complexity in their interaction with host proteins, and their de novo creation. This shows the ability of begomoviruses and their associated satellites to rapidly

[1] Amin, I., Patil, B., Briddon, R. W., Mansoor, S., and Fauquet, C. M. (2011). A common set of developmental miRNAs are upregulated in *Nicotiana benthamiana* by diverse

[2] Argüello-Astorga, G. R., and Ruiz, M. R. (2001). An iteron-related domain is associated to Motif 1 in the replication proteins of geminiviruses: identification of potential interacting amino acid-base pairs by a comparative approach. *Arch Virol* 146, 1465–1485. [3] Azzam, O., Frazer, J., de-la-Rosa, D., Beaver, J. S., Ahlquist, P., and Maxwell, D. P. (1994). Whitefly transmission and efficient ssDNA accumulation of *bean golden mosaic* 

[4] Bisaro, D. (2006). Silencing suppression by geminivirus proteins. *Virology* 344, 158 - 168. [5] Boulton, M. I., Steinkellner, H., Donson, J., Markham, P. G., King, D. I., and Davies, J. W. (1989). Mutational analysis of the virion-sense genes of *maize streak virus*. *J Gen Virol* 

[6] Briddon, R. W. (2003). Cotton leaf curl disease, a multicomponent begomovirus

[7] Briddon, R. W., Bull, S., Amin, I., Idris, A., Mansoor, S., Bedford, I., Dhawan, P., Rishi, N., Siwatch, S., and Abdel-Salam, A. (2003a). Diversity of DNA β: a satellite molecule

associated with some monopartite begomoviruses. *Virology* 312, 106 - 121.

GmusSA and GDarSLA act as a strong suppressor of PTGS.

evolve in response to selection pressures such as host plant resistance.

*NODAI Research Institute, Tokyo University of Agriculture, Tokyo, Japan* 

*Division of Crop Improvement, Directorate of Wheat Research, Karnal, India* 

*geminivirus* require functional coat protein. *Virology* 204(1), 289-96.

Muhammad Shafiq Shahid and Masato Ikegami

begomoviruses. *Virology Journal* 8(1), 143.

complex. *Molecular Plant Pathol*4, 427 - 434.

**9. Conclusions** 

**Author details** 

Pradeep Sharma \*

**10. References** 

70(Pt 9), 2309-23.

Corresponding Author

 \*

#### **8.1. Betasatellite**

Betasatellites have about 200nt sequences (known as a-Rich region) conserved among all that indicating may be these sequences have some biological roles in satellites. The role of A-Rich sequence may be to increase the required size of the molecule that is essential for encapsidation or systemic movement by the coat protein or movement protein encoded by begomovirus. TYLCCNB-Y10 could be infectious and mutant betasatellite (deleted a-rich region) could be encapsidated in the coat protein encoded by DNA-A that suggested may be A-Rich region is not required for trans-replication of TYLCCNB but only for size maintaining [95]. For example [20] reported that only a small region of the nucleotide sequence of CLCuMB upstream of the start codon of βC1 (a 68-nt fragment), which contains a G-box, was important for βC1 promoter activity. In addition to βC1 ORF of CLCuMB delete a larger region (complete βC1) to make it a gene delivery vector for plants. It can potentially tolerate the insertion of larger foreign sequences without affecting promoter activity [38]. Putative promoter and TATA box are located upstream of the *βC1* gene. Thus, the *βC1* gene of betasatellite could be replaced by a foreign gene and be modified to convert it into an expression vector [17]. The modified betasatellite might be an candidate gene silencing vector to study functional genomics in plants [54]. Also leaf curl symptoms in Nicotiana species can be brought by transgenic expression of the *βC1* gene of TYLCCNB that the severity of the symptoms parallels the level of βC1 transcript in the transgenic plants and their ability to induce symptoms is abolished by mutation of the *βC1* gene. Possibly *βC1* gene of betasatellite may be replaced with a foreign gene and used as an expression vector for gene function analysis in plants [20].

Evidence has been shown that TYLCCNB modified by deletion of its *βC1* gene but retaining the βC1 promoter and terminator, can be turned into a gene silencing vector. Also, insertion in the vector with fragments of endogenous plant genes or a transgene, in either the sense or antisense direction, can result in effective silencing of the cognate gene in plants [95]. The size of the mutated satellite DNA molecule significantly influences replication efficiency. TLCV sat-DNA can be used as a potential gene expression/silencing vector [47].

#### **8.2. Alphasatellite**

Also alphasatellite is a small molecule and easy to manipulate and have a wide host range and can apparently be maintained by a large number of distinct Begomovirus species. It has some sequences (A-rich approx.200 nt.), similar to betasatellite which can, potentially, be removed and still it can replicate autonomous. The A-rich deleted sequences of CLCuMA can not affect its ability to replicate autonomously and move, in trans, by a helper begomovirus that provide a space suitable for insertion of foreign sequences to increase its capacity to accept and maintain foreign gene sequences [82] (Table 2). This ability to amplify itself is useful for construction of VIGS vectors it will increase the copy number (and thus also expression) of inserted sequences [82]. Rolling-circle replication initiator protein of GmusSA and GDarSLA act as a strong suppressor of PTGS.

#### **9. Conclusions**

232 Plant Science

**vectors** 

**8.1. Betasatellite** 

for gene function analysis in plants [20].

**8.2. Alphasatellite** 

**8. Potential of mutated satellites using a virus induced gene silencing** 

Betasatellites have about 200nt sequences (known as a-Rich region) conserved among all that indicating may be these sequences have some biological roles in satellites. The role of A-Rich sequence may be to increase the required size of the molecule that is essential for encapsidation or systemic movement by the coat protein or movement protein encoded by begomovirus. TYLCCNB-Y10 could be infectious and mutant betasatellite (deleted a-rich region) could be encapsidated in the coat protein encoded by DNA-A that suggested may be A-Rich region is not required for trans-replication of TYLCCNB but only for size maintaining [95]. For example [20] reported that only a small region of the nucleotide sequence of CLCuMB upstream of the start codon of βC1 (a 68-nt fragment), which contains a G-box, was important for βC1 promoter activity. In addition to βC1 ORF of CLCuMB delete a larger region (complete βC1) to make it a gene delivery vector for plants. It can potentially tolerate the insertion of larger foreign sequences without affecting promoter activity [38]. Putative promoter and TATA box are located upstream of the *βC1* gene. Thus, the *βC1* gene of betasatellite could be replaced by a foreign gene and be modified to convert it into an expression vector [17]. The modified betasatellite might be an candidate gene silencing vector to study functional genomics in plants [54]. Also leaf curl symptoms in Nicotiana species can be brought by transgenic expression of the *βC1* gene of TYLCCNB that the severity of the symptoms parallels the level of βC1 transcript in the transgenic plants and their ability to induce symptoms is abolished by mutation of the *βC1* gene. Possibly *βC1* gene of betasatellite may be replaced with a foreign gene and used as an expression vector

Evidence has been shown that TYLCCNB modified by deletion of its *βC1* gene but retaining the βC1 promoter and terminator, can be turned into a gene silencing vector. Also, insertion in the vector with fragments of endogenous plant genes or a transgene, in either the sense or antisense direction, can result in effective silencing of the cognate gene in plants [95]. The size of the mutated satellite DNA molecule significantly influences replication efficiency.

Also alphasatellite is a small molecule and easy to manipulate and have a wide host range and can apparently be maintained by a large number of distinct Begomovirus species. It has some sequences (A-rich approx.200 nt.), similar to betasatellite which can, potentially, be removed and still it can replicate autonomous. The A-rich deleted sequences of CLCuMA can not affect its ability to replicate autonomously and move, in trans, by a helper begomovirus that provide a space suitable for insertion of foreign sequences to increase its capacity to accept and maintain foreign gene sequences [82] (Table 2). This ability to amplify itself is useful for construction of VIGS vectors it will increase the copy number (and thus

TLCV sat-DNA can be used as a potential gene expression/silencing vector [47].

The monopartite begomovirus associated with DNA-satellites (Betasatellite and Alphasatellite ) complex is in the norm throughout the Old World, particularly in South Asian countries. The epidemiology and evolution of this complex has been extensively analyzed since its first description. Monopartite begomovirus encoded all the genes needed to cause a successful infection. Many of these genes are coding for multifunctional proteins, adding another level of complexity in their interaction with host proteins, and their de novo creation. This shows the ability of begomoviruses and their associated satellites to rapidly evolve in response to selection pressures such as host plant resistance.

## **Author details**

Muhammad Shafiq Shahid and Masato Ikegami *NODAI Research Institute, Tokyo University of Agriculture, Tokyo, Japan* 

Pradeep Sharma \* *Division of Crop Improvement, Directorate of Wheat Research, Karnal, India* 

#### **10. References**


<sup>\*</sup> Corresponding Author

[8] Briddon, R. W., Bull, S. E., Amin, I., Mansoor, S., Bedford, I. D., Dhawan, P., Rishi, N., Siwatch, S. S., Zafar, Y., Abdel-Salam, A. M., and Markham, P.G. (2004). Diversity of DNA 1: a satellite molecule associated with some monopartite begomoviruses-DNA β complex. *Virology* 324(2),462-474.

Mutational Analysis of Effectors Encoded by Monopartite Begomoviruses and Their Satellites 235

[23] Etessami, P., Watts, J., and Stanley, J. (1989). Size reversion of *African cassava mosaic virus*  coat protein gene deletion mutants during infection of *Nicotiana benthamiana*. *J Gen Virol* 

[24] Frischmuth, T., and Stanley, J. (1998). Recombination between viral DNA and the transgenic coat protein gene of *African cassava mosaic geminivirus*. *J Gen Virol* 79(Pt 5),

[25] Gilbertson, R. L., Sudarshana, M., Jiang, H., Rojas, M. R., and Lucas, W. J. (2003). Limitations on geminivirus genome size imposed by plasmodesmata and virusencoded

[26] Gopal, P., Kumar, P., Sinilal, B., Jose, J., Kasin Yadunandam, A., and Usha, R. (2007). Differential roles of C4 and βC1 in mediating suppression of post-transcriptional gene silencing: Evidence for transactivation by the C2 of *bhendi yellow vein mosaic virus*, a

[27] Gorlich, D., and Kutay, U. (1999). Transport between the cell nucleus and the

[28] Hanley-Bowdoin, L., Settlage, S., and Robertson, D. (2004). Reprogramming plant gene expression: a prerequisite to geminivirus DNA replication. *Molcular Plant Pathol*5, 149 -

[29] Hanley-Bowdoin, L., Settlage, S. B., Orozco, B. M., Nagar, S., and Robertson, D. (1999). Geminiviruses: Models for Plant DNA Replication, Transcription, and Cell Cycle

[30] Heyraud-Nitschke, F., Schumacher, S., Laufs, J., Schaefer, S., Schell, J., and Gronenborn, B. (1995). Determination of the origin cleavage and joining domain of geminivirus Rep

[31] Ho, S. N., Hunt, H. D., Horton, R. M., Pullen, J. K., and Pease, L. R. (1989). Sitedirected mutagenesis by overlap extension using the polymerase chain reaction. *Gene* 77(1), 51-9. [32] Hofer, P., Engel, M., Jeske, H., and Frischmuth, T. (1997). Nucleotide sequence of a new bipartite geminivirus isolated from the common weed Sida rhombifolia in Costa Rica. *J* 

[33] Höhnle, M., Höfer, P., Bedford, I. D., Briddon, R. W., Markham, P. G., and Frischmuth, T. (2001). Exchange of three amino acids in the coat protein results in efficient whitefly transmission of a non transmissible *abutilon mosaic virus* isolate. *Virology* 290, 164-171. [34] Hong, Y., Stanley, J., and van Wezel, R. (2003). Novel system for the simultaneous analysis of geminivirus DNA replication and plant interactions in *Nicotiana benthamiana*.

[35] Idris, M. A., Shahid, M. S., Briddon, R. W., Khan, A. J., Zhu, J. K., and Brown, J. K. (2011). An unusual alphasatellite associated with monopartite begomoviruses attenuates symptoms and reduces betasatellite accumulation. *J Gen Virol* 92, 706–717. [36] Karpova, O. V., Ivanov, K. I., Rodionova, N. P., Dorokhov Yu, L., and Atabekov, J. G. (1997). Nontranslatability and dissimilar behavior in plants and protoplasts of viral

RNA and movement protein complexes formed in vitro. *Virology* 230(1), 11-21. [37] Karpova, O. V., Rodionova, N. P., Ivanov, K. I., Kozlovsky, S. V., Dorokhov, Y. L., and Atabekov, J. G. (1999). Phosphorylation of *Tobacco Mosaic Virus* Movement Protein

Abolishes Its Translation Repressing Ability. *Virology* 261(1), 20-24.

movement protein: insights into DNA trafficking. *Plant Cell* 15(11), 2578-91.

cytoplasm. *Annual Review of Cell and Development Biology* 15, 607-60.

Regulation. *Critical Reviews in Plant Sciences* 18(1), 71-106.

monopartite begomovirus. *Virus Res*123, 9 - 18.

proteins. *Nucleic Acids Research* 23, 910-916.

*Gen Virol* 78, 1785 - 1790.

*J Virology* 77(24), 13315-22.

70(Pt 2), 277-89.

1265-71.

156.


[23] Etessami, P., Watts, J., and Stanley, J. (1989). Size reversion of *African cassava mosaic virus*  coat protein gene deletion mutants during infection of *Nicotiana benthamiana*. *J Gen Virol*  70(Pt 2), 277-89.

234 Plant Science

30.

13966 - 13974.

1157-1170.

737-46.

complex. *Virology* 324(2),462-474.

vivo. *Virology* 409(2), 156-162.

nucleus. *J Virol*79, 10764 - 10775.

[8] Briddon, R. W., Bull, S. E., Amin, I., Mansoor, S., Bedford, I. D., Dhawan, P., Rishi, N., Siwatch, S. S., Zafar, Y., Abdel-Salam, A. M., and Markham, P.G. (2004). Diversity of DNA 1: a satellite molecule associated with some monopartite begomoviruses-DNA β

[9] Briddon, R. W., Mansoor, S., Bedford, I. D., Pinner, M. S., Saunders, K., Stanley, J., Zafar, Y., Malik, K. A., and Markham, P. G. (2001). Identification of DNA components

[10] Briddon, R. W., Pinner, M., Stanley, J., and Markham, P. (1990). Geminivirus coat

[11] Briddon, R. W., and Stanley, J. (2006). Sub-viral agents associated with plant-infecting

[12] Briddon, R. W., Watts, J., Markham, P. G., and Stanley, J. (1989). The coat protein of *beet* 

[13] Cheng, X., Wang, X., Wu, J., Briddon, R. W., and Zhou, X. (2011). βC1 encoded by tomato yellow leaf curl China betasatellite forms multimeric complexes in vitro and in

[14] Clerot, D., and Bernardi, F. (2006). DNA helicase activity is associated with the replication initiator protein rep of *tomato yellow leaf curl geminivirus*. *J Virol*80(22), 11322-

[15] Cui, X., Li, G., Wang, D., Hu, D., and Zhou, X. (2005a). A begomovirus DNA betaencoded protein binds DNA, functions as a suppressor of RNA silencing, and

[16] Cui, X., Li, G., Wang, D., Hu, D., and Zhou, X. (2005b). A begomovirus DNAβ-encoded protein binds DNA, functions as a suppressor of RNA silencing, and targets the cell

[17] Cui, X., Tao, X., Xie, Y., Fauquet, C. M., and Zhou, X. (2004). A DNAbeta associated with *tomato yellow leaf curl China* virus is required for symptom induction. *J Virol*78,

[18] Dry, I. B., Leslie, R., Krake, Justin, E., Rigden, and Rezaian, M. A. (1997). A novel subviral agent associated with a geminivirus: The first report of a DNA satellite.

[19] Eagle, P. A., Orozco, B. M., and Hanley-Bowdoin, L. (1994). A DNA sequence required for geminivirus replication also mediates transcriptional regulation. *Plant Cell* 6(8),

[20] Eini, O., Akbar Behjatnia, S. A., Satish, D., Dry, I. B., Randles, J. W., and Rezaian, M. A. (2009a). Identification of sequence elements regulating promoter activity and replication of a monopartite begomovirus-associated DNA beta satellite. *J Gen Virol* 90, 253–260. [21] Eini, O., Dogra, S., Selth, L. A., Dry, I. B., Randles, J. W., and Rezaian, M. A. (2009b).Interaction with a host ubiquitin-conjugating enzyme is required for the pathogenicity of a geminiviral DNA β satellite. *Molecular Plant Microbe Interaction* 22(6),

[22] Elmer, J. S., Brand, L., Sunter, G., Gardiner, W. E., Bisaro, D. M., and Rogers, S. G. (1988). Genetic analysis of the *tomato golden mosaic virus*. II. The product of the AL1 coding sequence is required for replication. *Nucleic Acids Research* 16(14B), 7043-60.

required for induction of cotton leaf curl disease. *Virology* 285, 234 - 243.

protein replacement alters insect specificity. *Virology* 177, 85 - 94.

*curly top virus* is essential for infectivity. *Virology* 172(2), 628-633.

*Proceedings of the National Academy of Sciences* 94(13), 7088-7093.

single-stranded DNA viruses. *Virology* 344, 198 - 210.

targets the cell nucleus. *J Virol*79, 10764 - 10775.


[38] Kharazmi, S., Behjatnia, S. A., Hamzehzarghani, H., and Niazi, A. (2012). Cotton leaf curl Multan betasatellite as a plant gene delivery vector trans-activated by taxonomically diverse geminiviruses. *Arch Virol* 5, 5.

Mutational Analysis of Effectors Encoded by Monopartite Begomoviruses and Their Satellites 237

disease is associated with multiple monopartite begomoviruses supported by single

[54] Mansoor, S., Briddon, R. W., Zafar, Y., and Stanley, J. (2003b). Geminivirus disease

[55] Mansoor, S., Khan, S. H., Bashir, A., Saeed, M., Zafar, Y., Malik, K., Briddon, R. W., Stanley, J., and Markham, P. G. (1999a). Identification of a novel circular single-stranded

DNA associated with cotton leaf curl disease in Pakistan. *Virology* 259, 190 -199. [56] Mansoor, S., Khan, S. H., Bashir, A., Saeed, M., Zafar, Y., Malik, K. A., Briddon, R. W., Stanley, J., and Markham, P. G. (1999b). Identification of a novel circular singlestranded

DNA associated with cotton leaf curl disease in Pakistan. *Virology* 259, 190 -199. [57] Meister, G., and Tuschl, T. (2004). Mechanisms of gene silencing by double-stranded

[58] Mubin, M., Amin, I., Amrao, L., Briddon, R. W., and Mansoor, S. (2010). The hypersensitive response induced by the V2 protein of a monopartite begomovirus is

[59] Nawaz-ul-Rehman, M. S., Mansoor, S., Briddon, R. W., and Fauquet, C. M. (2009). Maintenance of an Old World betasatellite by a New World helper begomovirus and

[60] Nawaz-ul-Rehman, M. S., Nahid, N., Mansoor, S., Briddon, R. W., and Fauquet, C. M. (2010). Post-transcriptional gene silencing suppressor activity of the alpha-Rep of nonpathogenic alphasatellites associated with begomoviruses. *Virology* 405, 300 -308. [61] Noris, E., Jupin, I., Accotto, G. P., and Gronenborn, B. (1996). DNA-binding activity of

[62] Noris, E. V., A. M. Caciagli, P. Masenga, V. Gronenborn, B. Accotto,G. P. (1998). Amino acids in the capsid protein of *tomato yellow leaf curl virus* that are crucial for systemic

[64] Padidam, M., Beachy, R. N., and Fauquet, C. M. (1996). The role of AV2 ("precoat") and coat protein in viral replication and movement in *tomato leaf curl geminivirus*. *Virology* 

[65] Pascal, E., Goodlove, P. E., Wu, L. C., and Lazarowitz, S. G. (1993). Transgenic tobacco plants expressing the geminivirus BL1 protein exhibit symptoms of viral disease. *Plant* 

[66] Qazi, J., Amin, I., Mansoor, S., Iqbal, M. J., and Briddon, R. W. (2007). Contribution of the satellite encoded gene βC1 to cotton leaf curl disease symptoms. *Virus Res* 128(1-2),

[67] Qian, Y., and Zhou, X. (2005). Pathogenicity and stability of a truncated DNAβ

[68] Rhee, Y., Gurel, F., Gafni, Y., Dingwall, C., and Citovsky, V. (2000). A genetic system for detection of protein nuclear import and export. *Nature Biotechlogy* 18(4), 433-437. [69] Rigden, J. E., Dry, I. B., Mullineaux, P. M., and Rezaian, M. A. (1993). Mutagenesis of the virion-sense open reading frames of *tomato leaf curl geminivirus*. *Virology* 193(2),1001-1005.

associated with *tomato yellow leaf curl China* virus. *Virus Res* 109(2), 159-163.

infection, particle formation, and insect transmission. *J Virol*72 (12 ), 10050-10057. [63] Orozco, B. M., Kong, L.-J., Batts, L. A., Elledge, S., and Hanley-Bowdoin, L. (2000). The multifunctional character of a geminivirus replication protein is reflected by its complex

DNA β. *Arch Virol* 148(10), 1969-1986.

RNA. *Nature* 431(7006), 343-349.

224, 390 - 404.

*Cell* 5(7), 795-807.

135-139.

complexes: an emerging threat. *Trends Plant Sci* 8, 128 - 134.

countered by the C2 protein. *Mol Plant Pathol* 11(2), 245–254.

oligomerization properties. *J Biol Chem* 275, 6114-6122.

possible rapid adaptation of the betasatellite. *J Virol* 83, 9347 - 9355.

the C2 protein of *tomato yellow leaf curl geminivirus*. *Virology* 217(2), 607-612.


disease is associated with multiple monopartite begomoviruses supported by single DNA β. *Arch Virol* 148(10), 1969-1986.

[54] Mansoor, S., Briddon, R. W., Zafar, Y., and Stanley, J. (2003b). Geminivirus disease complexes: an emerging threat. *Trends Plant Sci* 8, 128 - 134.

236 Plant Science

[38] Kharazmi, S., Behjatnia, S. A., Hamzehzarghani, H., and Niazi, A. (2012). Cotton leaf curl Multan betasatellite as a plant gene delivery vector trans-activated by

[39] Kheyr-Pour, A. B., M. Matzeit, V. Accotto, G. P. Crespi, S. Gronenborn, B. (1991). *tomato yellow leaf curl virus* from Sardinia is a whitefly-transmitted monopartite geminivirus.

[40] Kon, T., Rojas, M., Abdourhamane, I., and Gilbertson, R. (2009). Roles and interactions of begomoviruses and satellite DNAs associated with Okra leaf curl disease in Mali,

[41] Kon, T., Sharma, P., and Ikegami, M. (2007). Suppressor of RNA silencing encoded by the monopartite *tomato leaf curl Java begomovirus*. *Arch Virol* 152(7), 1273-1282. [42] Kumar P, P., Usha, R., Zrachya, A., Levy, Y., Spanov, H., and Gafni, Y. (2006). Protein– protein interactions and nuclear trafficking of coat protein and βC1 protein associated

[43] Kunik, T., Palanichelvam, K., Czosnek, H., Citovsky, V., and Gafni, Y. (1998). Nuclear import of the capsid protein of *tomato yellow leaf curl virus* (TYLCV) in plant and insect

[44] Lacatus, G., and Sunter, G. (2008). Functional analysis of bipartite begomovirus coat

[45] Laufs, J., Traut, W., Heyraud, F., Matzeit, V., Rogers, S. G., Schell, J., and Gronenborn, B. (1995). *In vitro* cleavage and ligation at the viral origin of replication by the replication

[46] Lazarowitz, S. G., Pinder, A. J., Damsteegt, V. D., and Rogers, S. G. (1989). *Maize streak virus* genes essential for systemic spread and symptom development. *EMBO J* 8(4),

[47] Li, D., Akbar Behjatnia, S. A., Dry, I. B., Randles, J. W., Eini, O., and Rezaian, M. A. (2007). Genomic regions of *tomato leaf curl virus* DNA satellite required for replication and for satellite-mediated delivery of heterologous DNAs. *J Gen Virol* 88(7), 2073-2077. [48] Lin, B., Akbar Behjatnia, S. A., Dry, I. B., Randles, J. W., and Rezaian, M. A. (2003). High-Affinity Rep-Binding Is not Required for the Replication of a Geminivirus DNA

[49] Liu, H., Boulton, M. I., Oparka, K. J., and Davies, J. W. (2001). Interaction of the movement and coat proteins of *maize streak virus*: implications for the transport of viral

[50] Liu, H., Boulton, M. I., Thomas, C. L., Prior, D. A., Oparka, K. J., and Davies, J. W. (1999). *Maize streak virus* coat protein is karyophyllic and facilitates nuclear transport of

[51] Liu, Y., Robinson, D. J., and Harrison, B. D. (1998). Defective forms of *cotton leaf curl virus* DNA-A that have different combinations of sequence deletion, duplication,

[52] Llave, C., Kasschau, K. D., Rector, M. A., and Carrington, J. C. (2002). Endogenous and Silencing-Associated Small RNAs in Plants. *The Plant Cell Online* 14(7), 1605-1619. [53] Mansoor, S., Briddon, R. W., Bull, S. E., Bedford, I. D., Bashir, A., Hussain, M., Saeed, M., Zafar, Y., Malik, K. A., Fauquet, C. M., and Markham, P. G. (2003a). Cotton leaf curl

with Bhendi yellow vein mosaic disease. *Virus Res*122(1–2), 127-136.

taxonomically diverse geminiviruses. *Arch Virol* 5, 5.

protein promoter sequences. *Virology* 376(1), 79-89.

and Its Satellite. *Virology* 305(2), 353-363.

viral DNA. *Mol Plant Microbe Interact* 12(10), 894-900.

inversion and rearrangement. *J Gen Virol* 79(P6), 1501-8.

DNA. *J Gen Virol* 82(1), 35-44.

protein of *tomato yellow leaf curl virus*. *PNAS* 92, 3879-3883.

*Nucl Acids Res* 19(24), 6763–6769.

cells. *Plant J* 13(3), 393-9.

1023-32.

West Africa. *J Gen Virol* 90, 1001 - 1013.


[70] Rojas, M. R., Jiang, H., Salati, R., Xoconostle-Cazares, B., Sudarshana, M.R., Lucas, W.J., Gilbertson, R.L. (2001). Functional analysis of proteins involved in movement of the monopartite begomovirus, *tomato yellow leaf curl virus*. *Virology* 291, 110–125.

Mutational Analysis of Effectors Encoded by Monopartite Begomoviruses and Their Satellites 239

[85] Stanley, J., Bisaro, D. M., Briddon, R. W., Brown, J. K., Fauquet, C. M., Harrison, B. D., Rybicki, E. P., and Stenger, D. C. (2005a). Geminiviridae. *Virus Taxonomy, VIIIth Report of* 

[86] Stanley, J., Bisaro, D. M., Briddon, R. W., Brown, J. K., Fauquet, C. M., Harrison, B. D., Rybicki, E. P., and Stenger, D. C. (2005b). Geminiviridaea. *Virus Taxonomy, VIIIth Report* 

[87] Stanley, J., Bisaro, D. M., Briddon, R. W., Brown, J. K., Fauquet, C. M., Harrison, B. D., Rybicki, E. P., Stenger, D. C., by, E., Fauquet, C. M., Mayo, M. A., Maniloff, J., Desselberger, J., and Ball, L. A. (2005c). Geminiviridae. In Virus Taxonomy, 5th report

[88] Stanley, J., and Latham, J. R. (1992). A symptom variant of *beet curly top geminivirus* 

[89] Stanley, J., Saunders, K., Pinner, M. S., and Wong, S. M. (1997). Novel defective interfering dnas associated with *ageratum yellow vein geminivirus* infection of ageratum

[90] Sunter, G., and Bisaro, D. (1997). Regulation of a geminivirus coat protein promoter by AL2 protein (TrAP): evidence for activation and derepression mechanisms. *Virology* 232,

[91] Sunter, G., and Bisaro, D. M. (1991). Transactivation in a geminivirus: AL2 gene product

[92] Sunter, G., Hartitz, M. D., Hormuzdi, S. G., Brough, C. L., and Bisaro, D. M. (1990). Genetic analysis of *tomato golden mosaic virus*: ORF AL2 is required for coat protein accumulation

[93] Sunter, G., Stenger, D. C., and Bisaro, D. M. (1994). Heterologous complementation by

[94] Talya Kunik, L. M., Vitaly Citovsky and Yedidya Gafni (1999). Characterization of a tomato karyopherin α that interacts with the *tomato yellow leaf curl virus* (TYLCV)

[95] Tao, X., Qing, L., and Zhou, X. (2004). Function of A-Rich region in DNAβ associated

[96] Tao, X., and Zhou, X. (2004). A modified viral satellite DNA that suppresses gene

[97] Trinks, D. R., R. Shivaprasad, P. V. Akbergenov, R. Edward J. Oakeley, K. Veluthambi, Thomas Hohn, Pooggin, M. M. (2005). Suppression of RNA silencing by a *geminivirus*  nuclear protein, ac2, correlates with transactivation of host genes. *J Virol* 79(4), 2517-2527. [98] Van Wezel, R., Dong, X., Liu, H., Tien, P., Stanley, J., and Hong, Y. (2002). Mutation of three cysteine residues in *tomato yellow leaf curl China virus* C2 protein causes dysfunction in pathogenesis and posttranscriptional gene silencing suppression. *Mol*

[99] Vanitharani, R., Chellappan, P., Pita, J. S., and Fauquet, C. M. (2004). Differential roles of AC2 and AC4 of cassava geminiviruses in mediating synergism and suppression of

[100] Voinnet, O. (2005). Induction and suppression of RNA silencing: insights from viral

while ORF AL3 is necessary for efficient DNA replication. *Virology* 179(1), 69-77.

with *tomato yellow leaf curl China* virus. *Chinese Sci Bull* 49(14), 1490-1493.

produced by mutation of open reading frame C4. *Virology* 190(1), 506-509.

of the ICTV. *London: Elsevier/Academic Press*, pp 301–326.

is needed for coat protein expression. *Virology* 180(1), 416-9.

geminivirus AL2 and AL3 genes. *Virology* 203(2), 203-10.

posttranscriptional gene silencing. *J Virol*78, 9487 - 9498.

expression in plants. *Plant J* 38(5), 850-60.

*Plant Microbe Interaction* 15, 203 - 208.

infections. *Nature Rev Genet* 6(3), 206-220.

capsid protein. *Journal of Experimental Botany* 50(334), 731-732.

*the ICTV*, 301 - 326.

*of the ICTV*, 301 - 326.

269 - 280.

conyzoides. *Virology* 239(1), 87-96.


[85] Stanley, J., Bisaro, D. M., Briddon, R. W., Brown, J. K., Fauquet, C. M., Harrison, B. D., Rybicki, E. P., and Stenger, D. C. (2005a). Geminiviridae. *Virus Taxonomy, VIIIth Report of the ICTV*, 301 - 326.

238 Plant Science

[70] Rojas, M. R., Jiang, H., Salati, R., Xoconostle-Cazares, B., Sudarshana, M.R., Lucas, W.J., Gilbertson, R.L. (2001). Functional analysis of proteins involved in movement of the

[71] Ruiz, M. T., Voinnet, O., and Baulcombe, D. C. (1998). Initiation and Maintenance of

[72] Saeed, M., Behjatnia, S., Mansoor, S., Zafar, Y., Hasnain, S., and Rezaian, M. (2005). A single complementary-sense transcript of a geminiviral DNA β satellite is determinant

[73] Saeed, M., Mansoor, S., Rezaian, M. A., Briddon, R. W., and Randles, J. W. (2008). Satellite DNA beta overrides the pathogenicity phenotype of the C4 gene of *tomato leaf curl virus*, but does not compensate for loss of function of the coat protein and V2 genes.

[74] Saeed, S., Zafar, Y., Randles, J. W., and Rezaian, M. A. (2007). A monopartite begomovirus-associated DNA beta satellite substitutes for the DNA B of a bipartite

[75] Sanderfoot, A. A., and Lazarowitz, S. G. (1995). Cooperation in Viral Movement: The Geminivirus BL1 Movement Protein Interacts with BR1 and Redirects It from the

[76] Saunders, K., Bedford, I., Briddon, R., Markham, P., Wong, S., and Stanley, J. (2000). A unique virus complex causes Ageratum yellow vein disease. *PNAS* 97, 6890 - 6895. [77] Saunders, K., Bedford, I. D., and Stanley, J. (2001). Pathogenicity of a natural recombinant associated with ageratum yellow vein disease: implications for

[78] Saunders, K., Briddon, R. W., and Stanley, J. (2008). Replication promiscuity of DNAbetasatellites associated with monopartite begomoviruses; deletion mutagenesis of the *ageratum yellow vein virus* DNA β satellite localises sequences involved in

[79] Saunders, K., Norman, A., Gucciardo, S., and Stanley, J. (2004). The DNA beta satellite component associated with ageratum yellow vein disease encodes an essential

[80] Saunders, K., and Stanley, J. (1999). A nanovirus-like DNA component associated with yellow vein disease of Ageratum conyzoides: evidence for interfamilial recombination

[81] Settlage, S. B., See, R. G., and Hanley-Bowdoin, L. (2005). Geminivirus C3 protein:

[82] Shahid, M. S., Ali, L., Andleeb, S. (2009). The function of the a-rich region of the alphasatellite associated with the cotton leaf curl disease in Pakistan. *EurAsia J BioSci* 3,

[83] Sharma, A. M., A. Osaki, T. Ikegami, M. (1998). Characterization of virus-specific DNA forms from tomato tissues infected by *tobacco leaf curl virus*: evidence for a single genomic component producing defective DNA molecules. *Plant Pathol* 47, 787-793. [84] Sharma, P., Ikegami, M., and Kon, T. (2010). Identification of the virulence factors and suppressors of posttranscriptional gene silencing encoded *by ageratum yellow vein virus*,

replication enhancement and protein interactions. *J Virol* 79, 9885 - 9895.

begomovirus to permit systemic infection. *J Gen Virol* 88, 2881 - 2889.

geminivirus evolution and disease aetiology. *Virology* 282(1), 38-47.

monopartite begomovirus, *tomato yellow leaf curl virus*. *Virology* 291, 110–125.

Virus-Induced Gene Silencing. *Plant Cell* 10(6), 937-946.

of pathogenicity. *Mol Plant-Microbe Interact* 18, 7 - 14.

Nucleus to the Cell Periphery. *Plant Cell* 7(8), 1185–1194.

*Arch Virol* 153, 1367 - 1372.

replication. *J Gen Virol* 89, 3165 - 3172.

152-156.

pathogenicity protein (βC1). *Virology* 324, 37 - 47.

between plant DNA viruses. *Virology* 264, 142 - 152.

a monopartite begomovirus. *Virus Res* 149(1), 19-27.


[101] Von Arnim, A., Frischmuth, T., and Stanley, J. (1993). Detection and possible functions of *African cassava mosaic virus* DNA B gene products. *Virology* 192(1), 264-72.

**Chapter 10** 

© 2012 Atak and Çelik, licensee InTech. This is an open access chapter distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2012 Atak and Çelik, licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

**Micropropagation of** *Anthurium spp.*

Micropropagation as an alternative method to conventional propagation, the culture of somatic cells, tissues and organs of plants under controlled conditions is a suitable way to produce a large number of progeny plants which are genetically identical to the stock plant in a short time. The important property of the plant cells is totipotency which is a capacity to produce the whole plant from different plant parts. Micropropagation has some features to be chosen in commercial production such as multiplicative capacity in a relatively short time, healthy and disease-free production capacity and ability to generate population during

The genetic pattern of the plant is key element to select the propagation method. Using micropropagation techniques in plant biotechnology applications are costlier than conventional propagation methods. Propagation by using *in vitro* techniques instead of conventional methods offer some advantages like utilizing small pieces of plants called as explants to maintain the whole plant and increase their number. The main point is to evolve new strategies to lower the time and cost consumed per plant [2,3]. In tissue culture applications selection of initiating material is important in the beginning of the culture. Therefore it is easy to provide virus-free clones in a short time. Production of plants during all year long independent of seasonal changes, long storage periods make micropropagation preferable to propagate plants in short time. There are also some disadvantages of micropropagation. Adaptation of cultured plants to the environmental conditions need transitional period to allow the plants to produce organic matter by photosynthesis [2,4].

The main methods of *in vitro* propagation can be classified in two groups:

2. Propagation by the formation of adventitious shoots or adventitious somatic embryos

The meristem and shoot tip cultures are used to establish virus-free plant culture. Many important horticulture crops were propagated by meristem culture for rapid growth and

1. Propagation from axillary or terminal buds

Çimen Atak and Özge Çelik

http://dx.doi.org/10.5772/51426

**1. Introduction** 

a year [1-5].

Additional information is available at the end of the chapter


## **Micropropagation of** *Anthurium spp.*

Çimen Atak and Özge Çelik

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/51426

## **1. Introduction**

240 Plant Science

158(1–2), 8-11.

159 - 165.

1239.

silencing. *Virology* 396, 85-93.

*Protoplasma* 248. 281-288.

[101] Von Arnim, A., Frischmuth, T., and Stanley, J. (1993). Detection and possible functions

[102] Wang, M. B., X. Wu, L. Liu, L. Smith, N. A. Isenegger, D. Wu, R. Masuta, C. Vance, V. B. Watson, J. Rezaian M.A. (2004). On the role of RNA silencing in the pathogenicity

[103] Wartig, L., Kheyr-Pour, A., Noris, E., De Kouchkovsky, F., Jouanneau, F., Gronenborn, B., and Jupin, I. (1997). Genetic analysis of the monopartite *tomato yellow leaf curl geminivirus*: roles of V1, V2, and C2 orfs in viral pathogenesis. *Virology* 228(2), 132-140. [104] Woolston, C. J., Reynolds, H. V., Stacey, N. J., and Mullineaux, P. M. (1989). Replication of wheat dwarf virus DNA in protoplasts and analysis of coat protein

[105] Wu, P., and Zhou, X. (2005). Interaction between a nanovirus-like component and the

[106] Yaakov, N., Levy, Y., Belausov, E., Gaba, V., Lapidot, M., and Gafni, Y. (2011). Effect of a single amino acid substitution in the NLS domain of *tomato yellow leaf curl virus*-Israel (TYLCV-IL) capsid protein (CP) on its activity and on the virus life cycle. *Virus Res* 

[107] Zhang, S. C., Wege, C., and Jeske, H. (2001). Movement proteins (BC1 and BV1) of *abutilon mosaic geminivirus* are cotransported in and between cells of sink but not of source leaves as detected by green fluorescent protein tagging. *Virology* 290(2), 249-60. [108] Zhou, X., Xie, Y., Tao, X., Zhang, Z., Li, Z., and Fauquet, C. M. (2003). Characterization of DNAbeta associated with begomoviruses in China and evidence for co-evolution

[109] Zrachya, A., Glick, E., Levy, Y., Arazi, T., Citovsky, V., and Gafni, Y. (2006). Suppressor of RNA silencing encoded by *tomato yellow leaf curl virus*-Israel. *Virology* 358,

[110] Kon, T., Hidayat, S.H., Hase, S., Takahashi, H., and Ikegami, M. (2006). The natural occurrence of two distinct begomoviruses associated with DNAβ and a recombinant

[111] Ito, T., Kimbara,J., Sharma,P., and Ikegami, M. (2009). Interaction of tomato yellow leaf curl virus with diverse betasatellites enhances symptom severity. *Arch Virol* 154, 1233-

[112] Ogawa, T., Sharma, P. and Ikegami, M. (2008). The begomoviruses Honeysuckle yellow vein mosaic virus and Tobacco leaf curl Japan virus with DNAβ satellites cause

[113] Sharma, P., and Ikegami, M. (2009). Characterization of signals that dicate nuclear/nucleolar and cytoplasmic shuttling of the capsid protein of Tomato leaf curl

[114] Sharma, P., and Ikegami, M. (2010). Tomato leaf curl virus V2 protein is a determinant of virulence, hypersensitive response and suppression of posttranscriptional gene

[115] Sharma, P., Gaur, R.K. and Ikegami, M. (2011). Subcellular localization of V2 protein of Tomato leaf curl Java virus by using green fluorescent protein and yeast hybrid system.

DNA in a tomato plant from Indonesia. *Phytopathology* 96(5), 517-525.

*tobacco curly shoot virus*/satellite complex. *Acta Biochim Biophys Sin* 37, 25 - 31.

of *African cassava mosaic virus* DNA B gene products. *Virology* 192(1), 264-72.

and evolution of viroids and viral satellites. *PNAS* 101(9), 3275-3280.

mutants in protoplasts and plants. *Nucl Acids Res* 17(15), 6029-6041.

with their cognate viral DNA-A. *J Gen Virol* 84, 237 - 247.

yellow dwarf disease of tomato. *Virus Res* 137(2), 235-244.

Java virus associated with DNAβ satellite. *Virus Res* 144, 145-153.

Micropropagation as an alternative method to conventional propagation, the culture of somatic cells, tissues and organs of plants under controlled conditions is a suitable way to produce a large number of progeny plants which are genetically identical to the stock plant in a short time. The important property of the plant cells is totipotency which is a capacity to produce the whole plant from different plant parts. Micropropagation has some features to be chosen in commercial production such as multiplicative capacity in a relatively short time, healthy and disease-free production capacity and ability to generate population during a year [1-5].

The genetic pattern of the plant is key element to select the propagation method. Using micropropagation techniques in plant biotechnology applications are costlier than conventional propagation methods. Propagation by using *in vitro* techniques instead of conventional methods offer some advantages like utilizing small pieces of plants called as explants to maintain the whole plant and increase their number. The main point is to evolve new strategies to lower the time and cost consumed per plant [2,3]. In tissue culture applications selection of initiating material is important in the beginning of the culture. Therefore it is easy to provide virus-free clones in a short time. Production of plants during all year long independent of seasonal changes, long storage periods make micropropagation preferable to propagate plants in short time. There are also some disadvantages of micropropagation. Adaptation of cultured plants to the environmental conditions need transitional period to allow the plants to produce organic matter by photosynthesis [2,4].

The main methods of *in vitro* propagation can be classified in two groups:


The meristem and shoot tip cultures are used to establish virus-free plant culture. Many important horticulture crops were propagated by meristem culture for rapid growth and

virus elimination. Adventitious shoots or adventitious somatic embryos are established directly or indirectly. Cultures are directly started with the excised explants from the mother plant tissues for organogenesis or embryogenesis. If shoots or embryos regenerate on previously formed callus or in cell culture, they are called as indirect organogenesis or embryogenesis [3,6,7].

Micropropagation of *Anthurium spp.* 243

micropropagation. Application of biotechnology on *in vitro* propagation of *Anthurium* is

*In vitro* propagation methods have several advantages over conventional propagation like flexible adjustment of factors affecting regeneration such as explant type, nutrient and plant growth regulator levels and conditions of the environment, production of clones in desired rate, continued production during seasonal changes using tissue culture methods also

The success of tissue culture is related to the correct choice of explants. Shoot or shoot tips and node cultures are the most commonly used culture types in micropropagation of plants. Explants from shoot tips and nodal stem segments are suitable for enhanced axillary branching. *Anthurium* micropropagation from axillary buds, shoot tip, lamina explants, node, petiole, and microcuttings have been successfully utilized [15-18]. Among these plant

The genotype of *Anthurium* plays an important role in *in vitro* propagation. The studies showed that different genotypes had different responses to the same culture conditions. For this reason, it is necessary to establish a suitable procedure for each varieties of *Anthurium*

Selection of explant type to induce callogenesis and orgonogenesis is important for plants. In direct and indirect orgonogenesis studies, using young leaf explants are important for the success of culture. Martin et al.[2003] observed higher number of shoots in the brown young lamina explants than young green lamina. Viégas et al.[2007] also indicated the importance of using new brown leaves for callus induction. Bejoy et al.[2008] reported that the explants excised from pale green leaves showed better callus development than pale brown leaves. Atak and Çelik [2009] also used young brown and green leaves of *Anthurium andreanum* to evaluate the effectiveness of callus formations. They achieved to decrease the callus formation time by using brown leaf explants and induced the callus formation percentage

The second important step in micropropagation is to obtain aseptic culture of plant material. Aseptic culture systems are effective to eradicate the bacterial, fungal and insect contaminants. The sterilization protocols used for different *Anthurium* explant sources were given in Table 1. NaOCl is the main disinfection material used in establishing aseptic culture conditions of *Anthurium*. NaOCl has been used for the concentrations differ from 1%-5% [Table 1]. The incubation times of the explants in sodium hypochloride showed differences due to its concentrations. There is also need to used extra disinfectant solutions to eradicate the fungal and bacterial contaminants. Benomyl [commercial name

parts, leaves are the most used explant source in *in vitro* culture of *Anthurium*.

that can be adapted to commercial production [3,4,19,20].

50% more than performed by green leaves.

**Establishing aseptic culture** 

important to increase the productivity of *Anthurium* [3, 6, 13].

**2.1. Tissue culture of** *Anthurium*

**Explant type** 

increase the multiplication rate of plants [14].

When propagation occurs via an indirect callus phase, the genetic identity of the progenies decreases. This is an important problem in commercial propagation to affect the uniformity of progenies. Callus formation also increases the somaclonal variation. Increasing of somaclonal variation incidence is a crucial result of long term period of callus growth. Origin of the callus also causes somaclonal variation.

Propagation from axillary or terminal buds is the most ensurable method to have the highest genetic stability during *in vitro* propagation of plants.

George et al. [2008] described five stages of micropopagation which are mother plant selection and preparation [Stage 0], *in vitro* culture establishment [Stage 1], shoot multiplication [Stage 2], rooting of microshoots [Stage 3] and acclimatization [Stage 4]. These stages are necessary for a successful micropropagation.

Establishing aseptic culture conditions can be classified as Stage 0 which contains presurface sterilization applications of explants to reduce contamination of stock plants. The success of Stage 2 depends on different factors such as plant species, cultivar or genotype, plant growth regulators, the ingredients of the medium and physical culture conditions. Stage 3 is responsible of rooting of microshoots. It depends on the factors given in Stage 2. Transplantation of rooted shoots to the environment is the main step of Stage 4. This is also the important part of micropropagation. Acclimatization needs to be well controlled to avoid loss of propagated plants [4,5].

## **2. Propagation of** *Anthurium*

The commercial production of ornamental pot plants has a great potential in international markets. In the global market, *Anthurium* cultivars with valued flowers are the second beside the Orchids among tropical cut flowers. *Anthurium* species and hybrids in Araceae family have an importance in monocotyledonous ornamental plants and they are commercially produced as cut flowers and potted plants in tropical and subtropical countries [2, 8-12].

The propagation rate of *Anthurium* by seeds is very low and it is not recommended. The cultivation has also been limited because of the inherent heterozygosity. The time between pollination and seed maturity and the development time take three years in a breeding program. To grow plants from seed may not provide a practical method of making new planting areas, in such circumstances vegetative propagation [stem cutting] seems the only way of multiplying a unique individual. Propagation method selection for a plant depends on its genetic potential and its intended use. Stem cutting methods are also not practical to propagate in large scale. Today *Anthurium* can be multiplied in large number by micropropagation. Application of biotechnology on *in vitro* propagation of *Anthurium* is important to increase the productivity of *Anthurium* [3, 6, 13].

### **2.1. Tissue culture of** *Anthurium*

*In vitro* propagation methods have several advantages over conventional propagation like flexible adjustment of factors affecting regeneration such as explant type, nutrient and plant growth regulator levels and conditions of the environment, production of clones in desired rate, continued production during seasonal changes using tissue culture methods also increase the multiplication rate of plants [14].

#### **Explant type**

242 Plant Science

embryogenesis [3,6,7].

Origin of the callus also causes somaclonal variation.

stages are necessary for a successful micropropagation.

avoid loss of propagated plants [4,5].

**2. Propagation of** *Anthurium*

countries [2, 8-12].

highest genetic stability during *in vitro* propagation of plants.

virus elimination. Adventitious shoots or adventitious somatic embryos are established directly or indirectly. Cultures are directly started with the excised explants from the mother plant tissues for organogenesis or embryogenesis. If shoots or embryos regenerate on previously formed callus or in cell culture, they are called as indirect organogenesis or

When propagation occurs via an indirect callus phase, the genetic identity of the progenies decreases. This is an important problem in commercial propagation to affect the uniformity of progenies. Callus formation also increases the somaclonal variation. Increasing of somaclonal variation incidence is a crucial result of long term period of callus growth.

Propagation from axillary or terminal buds is the most ensurable method to have the

George et al. [2008] described five stages of micropopagation which are mother plant selection and preparation [Stage 0], *in vitro* culture establishment [Stage 1], shoot multiplication [Stage 2], rooting of microshoots [Stage 3] and acclimatization [Stage 4]. These

Establishing aseptic culture conditions can be classified as Stage 0 which contains presurface sterilization applications of explants to reduce contamination of stock plants. The success of Stage 2 depends on different factors such as plant species, cultivar or genotype, plant growth regulators, the ingredients of the medium and physical culture conditions. Stage 3 is responsible of rooting of microshoots. It depends on the factors given in Stage 2. Transplantation of rooted shoots to the environment is the main step of Stage 4. This is also the important part of micropropagation. Acclimatization needs to be well controlled to

The commercial production of ornamental pot plants has a great potential in international markets. In the global market, *Anthurium* cultivars with valued flowers are the second beside the Orchids among tropical cut flowers. *Anthurium* species and hybrids in Araceae family have an importance in monocotyledonous ornamental plants and they are commercially produced as cut flowers and potted plants in tropical and subtropical

The propagation rate of *Anthurium* by seeds is very low and it is not recommended. The cultivation has also been limited because of the inherent heterozygosity. The time between pollination and seed maturity and the development time take three years in a breeding program. To grow plants from seed may not provide a practical method of making new planting areas, in such circumstances vegetative propagation [stem cutting] seems the only way of multiplying a unique individual. Propagation method selection for a plant depends on its genetic potential and its intended use. Stem cutting methods are also not practical to propagate in large scale. Today *Anthurium* can be multiplied in large number by The success of tissue culture is related to the correct choice of explants. Shoot or shoot tips and node cultures are the most commonly used culture types in micropropagation of plants. Explants from shoot tips and nodal stem segments are suitable for enhanced axillary branching. *Anthurium* micropropagation from axillary buds, shoot tip, lamina explants, node, petiole, and microcuttings have been successfully utilized [15-18]. Among these plant parts, leaves are the most used explant source in *in vitro* culture of *Anthurium*.

The genotype of *Anthurium* plays an important role in *in vitro* propagation. The studies showed that different genotypes had different responses to the same culture conditions. For this reason, it is necessary to establish a suitable procedure for each varieties of *Anthurium* that can be adapted to commercial production [3,4,19,20].

Selection of explant type to induce callogenesis and orgonogenesis is important for plants. In direct and indirect orgonogenesis studies, using young leaf explants are important for the success of culture. Martin et al.[2003] observed higher number of shoots in the brown young lamina explants than young green lamina. Viégas et al.[2007] also indicated the importance of using new brown leaves for callus induction. Bejoy et al.[2008] reported that the explants excised from pale green leaves showed better callus development than pale brown leaves. Atak and Çelik [2009] also used young brown and green leaves of *Anthurium andreanum* to evaluate the effectiveness of callus formations. They achieved to decrease the callus formation time by using brown leaf explants and induced the callus formation percentage 50% more than performed by green leaves.

#### **Establishing aseptic culture**

The second important step in micropropagation is to obtain aseptic culture of plant material. Aseptic culture systems are effective to eradicate the bacterial, fungal and insect contaminants. The sterilization protocols used for different *Anthurium* explant sources were given in Table 1. NaOCl is the main disinfection material used in establishing aseptic culture conditions of *Anthurium*. NaOCl has been used for the concentrations differ from 1%-5% [Table 1]. The incubation times of the explants in sodium hypochloride showed differences due to its concentrations. There is also need to used extra disinfectant solutions to eradicate the fungal and bacterial contaminants. Benomyl [commercial name


is Benlate], Cetrimite, gentamicin and streptomycin sulphate are effectively used for this aim [11,13,15,18,20,22].

Micropropagation of *Anthurium spp.* 245

is reduced to

and reduced by NH4+. But

and NH4+ differs for different plants

has a key role in

to NH4+ ratio

Culture medium influences the propagation efficiency in plant tissue culture applications. Organic compounds, vitamins and plant growth regulators are used to stimulate healthy growth. The rate of tissue growth and morphogenetic responses highly affected by the

There are several basal media such as Chu [N6] [24], Gamborg's B5 [25], Murashige and Skoog [MS] [26], Murashige and Tucker [MT] [27] and Nitsch and Nitsch [NN] [28]. These media are successfully used for establishing tissue cultures of different explants of various plants [22].

In plant tissue culture studies, different combinations of every medium based on different concentrations of macro and micronutrients have been used to develop efficient protocols. The rapid and efficient tissue culture protocols are important for micropropagation of

The success of plant tissue culture depends on the composition of the medium used. Different combinations of macronutrients as nitrogen, potassium, calcium, phosphorus, magnesium and sulphur and micronutrients [trace elements] as iron, nickel, chlorine,

Each plant species has its own medium composition or it should be improved for better results. The modifications can be made up in macro and micronutrients, sugar content, plant

MS media with some modifications have been frequently applied in tissue culture of *Anthurium*. The differences caused by using different concentrations of plant growth

Nitrogen is an essential macronutrient in plant life. It is an important component of proteins

ammonium [NH4+] after uptake. Plants have ability to use the reduced form of nitrogen for their metabolism. Nitrate uptake happens effectively in an acidic pH. But after nitrate uptake, the medium are becoming less acid. When ammonium uptake, it makes the medium more acidic. The pH of the plant culture media is important because in a buffered media, existence of both ions affects efficient nitrogen uptake. The form and the amount of nitrogen in media have significant effects on cell growth and differentiation. pH controlling in the media is not the only reason of using both ions, excessive ammonium ions are toxic to the

morphogenesis is being controlled by total amount of nitrogen in the medium and it needs

and different kinds of cultures. This situation implies that this ratio should be specifically

] is the main source of nitrogen. NO3-

manganase, zinc, boron, copper and molybdenum change the nature of the medium.

regulators in combination with MS organics used to obtain desired tissues [Table 2].

plants. Media containing high levels of NH4+ also inhibits chlorophyll synthesis [4].

and NH4+. Because of using optimum NH4+: NO3-

adjusted for each plant species and for different purposes. Changing the NO3-

growth regulators, vitamins and other nitrogen supplements.

It has been known that the root growth is induced by NO3-

morphogenesis, therefore the balance between NO3-

by small alterations affects differentiation and growth.

**Culture medium** 

features of nutrients included.

*Anthurium* as much as in other plants.

and nucleic acids. Nitrate [NO3-

both of NO3-

**Table 1.** Sterilization methods used in *Anthurium* tissue culture

#### **Culture medium**

244 Plant Science

aim [11,13,15,18,20,22].

*A.andreanum L.* Apical shoot

buds

Leaf and spadix Segments

Seeds from plant spadix

segments

**Table 1.** Sterilization methods used in *Anthurium* tissue culture

Separate fruits from spadix Isolate seeds

*A.andreanum*  cv Rubrun

*A.andreanum* Hort Lamina

is Benlate], Cetrimite, gentamicin and streptomycin sulphate are effectively used for this

**A. species Explant Source Sterilization method Reference** 

Leaf 70% ethanol [1 min]+gentamicin

[30 sec]+1.5% NaOCl containing two drops of Tween 20 [20 min]

Leaf 0.1% HgCl2 [19]

[30 min]+20% [v/v] commersial bleach [5% NaOCl,12 min]

Cetrimite [5min.] +NaOCl [5 min]+

[30-60 min]+1% pesticide solution

streptomycin sulphate [30 min] +5 times distilled water [5min each

1% NaOCl [10 min] + 2% NaOCl [5min] +80% alcohol [30s.] +5-6 times distilled water [5min each

Washing under running tap water

3% [v/v]NaOCl [15 min]+3 times

1% [v/v]NaOCl [20 min]+2 times

1%NaOCl [20 min] [7]

[30 min]+0.5% [v/v]Trix [Commercial detergent]+70% ethanol [1 min.] + 1.5% NaOCl containing 0.01% Tween 20 [8 min]

distilled water [5min.]

distilled water [10min.]

chloride [10-12 min]

min]+0.1%HgCl2 [7 min]

Leaf 15% [v/v] commersial bleach [20

5% [v/v] Extran [5 min. with detergent]+0.1% [w/v] mercuric

Teepol+ antifungal solution

0.1% [w/v] HgCl2 [5min.]

of 50% benomyl and 20%

Spadices Washing under running tap water

rinse.]+

rinse.]

[15]

[20]

[11]

[22]

[23]

[18]

[1]

[21]

*A.andreanum* Leaf 0.6% Benlate [30 min]+70% ethanol

Culture medium influences the propagation efficiency in plant tissue culture applications. Organic compounds, vitamins and plant growth regulators are used to stimulate healthy growth. The rate of tissue growth and morphogenetic responses highly affected by the features of nutrients included.

There are several basal media such as Chu [N6] [24], Gamborg's B5 [25], Murashige and Skoog [MS] [26], Murashige and Tucker [MT] [27] and Nitsch and Nitsch [NN] [28]. These media are successfully used for establishing tissue cultures of different explants of various plants [22].

In plant tissue culture studies, different combinations of every medium based on different concentrations of macro and micronutrients have been used to develop efficient protocols. The rapid and efficient tissue culture protocols are important for micropropagation of *Anthurium* as much as in other plants.

The success of plant tissue culture depends on the composition of the medium used. Different combinations of macronutrients as nitrogen, potassium, calcium, phosphorus, magnesium and sulphur and micronutrients [trace elements] as iron, nickel, chlorine, manganase, zinc, boron, copper and molybdenum change the nature of the medium.

Each plant species has its own medium composition or it should be improved for better results. The modifications can be made up in macro and micronutrients, sugar content, plant growth regulators, vitamins and other nitrogen supplements.

MS media with some modifications have been frequently applied in tissue culture of *Anthurium*. The differences caused by using different concentrations of plant growth regulators in combination with MS organics used to obtain desired tissues [Table 2].

Nitrogen is an essential macronutrient in plant life. It is an important component of proteins and nucleic acids. Nitrate [NO3- ] is the main source of nitrogen. NO3 is reduced to ammonium [NH4+] after uptake. Plants have ability to use the reduced form of nitrogen for their metabolism. Nitrate uptake happens effectively in an acidic pH. But after nitrate uptake, the medium are becoming less acid. When ammonium uptake, it makes the medium more acidic. The pH of the plant culture media is important because in a buffered media, existence of both ions affects efficient nitrogen uptake. The form and the amount of nitrogen in media have significant effects on cell growth and differentiation. pH controlling in the media is not the only reason of using both ions, excessive ammonium ions are toxic to the plants. Media containing high levels of NH4+ also inhibits chlorophyll synthesis [4].

It has been known that the root growth is induced by NO3 and reduced by NH4+. But morphogenesis is being controlled by total amount of nitrogen in the medium and it needs both of NO3 and NH4+. Because of using optimum NH4+: NO3 has a key role in morphogenesis, therefore the balance between NO3 and NH4+ differs for different plants and different kinds of cultures. This situation implies that this ratio should be specifically adjusted for each plant species and for different purposes. Changing the NO3 to NH4+ ratio by small alterations affects differentiation and growth.


Micropropagation of *Anthurium spp.* 247

[22]

[21]

[17]

[6]

to NH4+ is

**Medium components Aim Reference** 

Callus Shoot regeneration

Roots

Roots

shoots

induction

Embryo induction

proliferation

Callus [10]

Shoot induction [1]

Callus [35]

Multiple shoots

MS+4.44mM BAP+2.89mM GA3 Shoot induction [16]

½MS+0.44µM BA Multiple shoots

MS+0.5mg/l IAA+2g/l AC Roots

NWT+0.25mg/l 2,4-D +0.02mg/l NAA+1.5mg/l TDZ + 0.75 mg/l

NWT+ 0. 2mg/l NAA+1.0

½MS+0.54µM+NAA+0.93µM

¼MS+0.88µM BA+0.54µM NAA+0.46µM Kin

Leaf, petiole ½MS+0.90µM 2,4-D+8.88µM BA Callus

NH4NO3+18µM 2,4-D+6%

BAP, 6-benzylaminopurine; BA, N6-benzyladenine; 2,4-D, 2,4-dichlorophenoxyacetic acid, IAA, indole-3-acetic acid; IBA, indole-3-butyric acid; 2-iP, N6-[2-isopentenyl]adenine; Kin, kinetin; NAA, α-naphthalene acetic acid; TDZ, thidiazuron; AC, activated charcoal; AS, adenin sulphate; MS, Murashige Skoog [1962] medium; WM: Winarto-

66:34 at this medium. For this reason generally modified MS medium used at *Anthurium* organogenesis. The modifications of ammonium nitrate concentration have been studied at *Anthurium* media by researchers. Hamidah et al. [1997] used half-strength MS

BAP+1mg/l 2-iP

Leaf Modified MS+2.5 mM

sucrose

**Table 2.** *In vitro culture medium components for Anthurium cultivars (Modified from [22*]).

MS media used frequently for tissue culture of *Anthurium* and the ratio of NO3-

½MS+0.54µM NAA Roots

½MS+0.90µM 2,4-D+4.44µM BA Callus

MS+5.71mM NAA Roots

MS+0.5mg/l BAP Shoots

½MS+0.5mg/l 2,4-D+1mg/l BAP Adventitious

*Anthurium species* 

*A.andreanum* Lindl.cv.

*A.andreanum* 

*A.scherzerianum* 

Schott

*Hort* 

**Explant source**

Half anther culture

Nodal segments BAP

Kin

*A.scherzerianum* Leaf ½MS+0.08mg/l 2,4-D+1mg/l

Teixeria medium, NWM: New Winarto-Teixeria medium GA3, giberellic acid.

mg/lKIN

Lamina ½MS+1.11µM+BA+1.14µM IAA+0.46µM Kin

Leaf ¼MS+0.88µM BA+0.9µM 2,4- D+0.46µM Kin


*Anthurium species* 

*A*.*andreanum* André cv.

*Anthurium andreaum* cv Rubun

*A.andreanum* Lind.

**Explant source**

*A.andreanum* Leaf MS+2.2-4.4µM BA+0.9µM 2,4-D Adventitious

Leaf Modified Nitsch [200mg/l

+0.5mg/l BA

2,4-D+1mg/l BA

*Anthurium* ssp. Leaf ½MS+1mg/l BA+0.08mg/l 2,4-D Callus

BA

Leaf, petiole Modified Pietrik

BA

Microcutting

Apical shoot

from germinated seed

bud

2,4-D

**Medium components Aim Reference** 

Root Modified MS+2.2µM BA Multiple shoots [34]

NH4NO3] +1mg/l BA+0.1mg/l

Nitsch [720mg/l NH4NO3]

Nitsch [720mg/l NH4NO3] +1.0mg/l IBA+0.04% AC

Leaf ½MS+0.6mg/l 2,4-D+1mg/l BAP Callus

Seed MS+2mg/l BA+0.5mg/l NAA Callus

½MS+250mg/l NH4NO3+0.1mg/l

½MS+1mg/l IBA+0.04% AC Roots Leaf, spadix ¼MS+1mg/l BAP Multiple shoots [23] ¼MS+1mg/l IBA Roots

Petiol ½MS+0.1mg/l 2,4-D+0.5 mg/l BA Callus [36] ½MS+0.1mg/l 2,4-D+1.0 mg/l BA Shoot ½MS+0.5mg/l 2,4-D Root

½MS+1mg/l BA Callus

¼MS+1g/l AC Roots

MS+0.1mg/l NAA+0.25mg/l BAP Multiple apical

½MS[206 mg/l NH4NO3] +1mg/l

medium+0.36µM 2,4-D+4.4µM

MS+4.4µM BA+0.05µM NAA

MS+0.5mg/l BAP+60mg/l adenine sulphate

shoots

Callus initiation

Shoot development

Roots

induction

proliferation

induction

Shoot regeneration

shoots

Multiple shoots

multiplication

Callus [32]

Multiple shoots [7]

Shoot regeneration [33]

[15]

[20]

[18]

[19]

[11]

BAP, 6-benzylaminopurine; BA, N6-benzyladenine; 2,4-D, 2,4-dichlorophenoxyacetic acid, IAA, indole-3-acetic acid; IBA, indole-3-butyric acid; 2-iP, N6-[2-isopentenyl]adenine; Kin, kinetin; NAA, α-naphthalene acetic acid; TDZ, thidiazuron; AC, activated charcoal; AS, adenin sulphate; MS, Murashige Skoog [1962] medium; WM: Winarto-Teixeria medium, NWM: New Winarto-Teixeria medium GA3, giberellic acid.

**Table 2.** *In vitro culture medium components for Anthurium cultivars (Modified from [22*]).

MS media used frequently for tissue culture of *Anthurium* and the ratio of NO3 to NH4+ is 66:34 at this medium. For this reason generally modified MS medium used at *Anthurium* organogenesis. The modifications of ammonium nitrate concentration have been studied at *Anthurium* media by researchers. Hamidah et al. [1997] used half-strength MS macroelements with 2.5 mM ammonium nitrate for *in vitro* stock cultures. While Puchooa [2005] used 200 mg/L reduced ammonium nitrate concentration for callus culture, they increased the amount to 720 mg/L for regeneration. Dufour and Guérin [2005] used different compositions of NO3 and NH4 to evaluate the developmental results. According to their results, the ratio of 0.37 showed better plant growth and development. Atak and Çelik [2009] preferred to use half-strength MS salts with NH4NO3 lowered to 250 mg/L for shoot regeneration. Winarto et al. [2011] were improved a protocol for callus induction and plant regeneration and NWT-3 media contains 750 mg/l NH4NO3.

Micropropagation of *Anthurium spp.* 249

The most important effect of using AC to the medium is the rigorous decrease in the concentrations of plant growth regulators and other organic supplements. AC shows greater adsorptive capacity to phenolics commonly produced by wounded tissues, plant hormones like IAA, NAA, IBA, BA, kinetin, zeatin and other hormones [30,31]. The adsorptive property of AC changes with purity, pH and density [3]. The *Anthurium* seedlings propagated by Atak and Çelik [2009] were rooted in medium containing AC and given in

**Figure 1.** *In vitro* propagation of *Anthurium* cultivars [Arizona]. The shoots with root were growth

In plant propagation applications, subculturing has an importance to prolong the life of plants and expand the number of cultured seedlings. At *in vitro* propagation of *Anthurium andreanum* cultivars, the number of shoots per explants was increased subsequent subcultures. Atak and Çelik [2009] observed that shoot multiplication for two *Anthurium andreanum* cultivars Arizona and Sumi was increased in the next multiplication stage. At every subculture, shoot numbers regenerated form nodal explants gradually increased [Table 3]. Bejoy et al.[2008] reported that multiplication was enhanced in the next multiplication stage. They succeeded to increase the rate of shoot production in the second

**3. The importance of subculturing in micropropagation** 

inplant tissue culture medium with AC [20].

multiplication stage.

Figure 1.

In culture conditions, using synthetic chemicals with similar physiological activities as plant hormones have capabilities to induce plant growth as desired. Auxin and cytokinins are the most important hormones regulating growth and morphogenesis in plant tissue culture. Their combinative usage promote growth of calli, cell suspensions, root and shoot development and have capability to regulate the morphogenesis [4,29]. There are synthetic auxin and cytokinins beside naturals. Different combinations and concentrations of plant growth regulators such as 2,4-dichlorophenoxyacetic acid [2,4-D], naphthalene acetic acid [NAA], benzylaminopurine [BAP] and kinetin [Kin] were used to indicate callus formation from different kinds of explants of *Anthurium* cultivars. In preliminary studies, induction and regeneration of callus followed by shoot and root regeneration are the main steps of tissue culture of whole plants. As an important commercial plant, to develop a rapid and more effective tissue culture protocol to shorten the time is the main objective of *Anthurium* tissue culture [7,10, 22,23].

As given in Table 2, combination of 2,4-D and BA in culture media to induce callus initiation from leaf explants in different varieties of *Anthurium* is frequently used. Also, adding of BAP and 2-iP to the callus medium has been evaluated by different researchers. The concentrations of 2,4-D used in the callus medium is ranging from 0.08 mg/l to 1 mg/l 2,4-D. The BA concentrations are changing between 0.1 mg/l and 1 mg/l.

Micropropagated plants require a developed root system to resist the external environmental conditions. Rooting of the shoots take place *in vitro*. Therefore, determination of the appropriate auxin type and levels in the media required to promote rooting [4].

Activated charcoal [AC] is added to medium for promoting root growth [11, 13, 15, 19, 20]. AC is composed of carbon and it is often used in plant tissue culture to absorb gases and dissolved solids. It is not a growth regulator but it has an ability to modify medium composition [4].

There are several advantageous uses of charcoal on the type of culture. These are adsorption of secreted compounds from cultured tissues, decrements in the phenolic oxidations, pH changes of the medium to optimize for morphogenesis, prevention of unwanted callus growth, simulation of soil conditions because of the ability to promote root formation, capability to use in production of secondary plant products in culture conditions [4, 30].

The most important effect of using AC to the medium is the rigorous decrease in the concentrations of plant growth regulators and other organic supplements. AC shows greater adsorptive capacity to phenolics commonly produced by wounded tissues, plant hormones like IAA, NAA, IBA, BA, kinetin, zeatin and other hormones [30,31]. The adsorptive property of AC changes with purity, pH and density [3]. The *Anthurium* seedlings propagated by Atak and Çelik [2009] were rooted in medium containing AC and given in Figure 1.

248 Plant Science

tissue culture [7,10, 22,23].

rooting [4].

composition [4].

conditions [4, 30].

macroelements with 2.5 mM ammonium nitrate for *in vitro* stock cultures. While Puchooa [2005] used 200 mg/L reduced ammonium nitrate concentration for callus culture, they increased the amount to 720 mg/L for regeneration. Dufour and Guérin [2005] used different compositions of NO3 and NH4 to evaluate the developmental results. According to their results, the ratio of 0.37 showed better plant growth and development. Atak and Çelik [2009] preferred to use half-strength MS salts with NH4NO3 lowered to 250 mg/L for shoot regeneration. Winarto et al. [2011] were improved a protocol for callus induction and plant

In culture conditions, using synthetic chemicals with similar physiological activities as plant hormones have capabilities to induce plant growth as desired. Auxin and cytokinins are the most important hormones regulating growth and morphogenesis in plant tissue culture. Their combinative usage promote growth of calli, cell suspensions, root and shoot development and have capability to regulate the morphogenesis [4,29]. There are synthetic auxin and cytokinins beside naturals. Different combinations and concentrations of plant growth regulators such as 2,4-dichlorophenoxyacetic acid [2,4-D], naphthalene acetic acid [NAA], benzylaminopurine [BAP] and kinetin [Kin] were used to indicate callus formation from different kinds of explants of *Anthurium* cultivars. In preliminary studies, induction and regeneration of callus followed by shoot and root regeneration are the main steps of tissue culture of whole plants. As an important commercial plant, to develop a rapid and more effective tissue culture protocol to shorten the time is the main objective of *Anthurium*

As given in Table 2, combination of 2,4-D and BA in culture media to induce callus initiation from leaf explants in different varieties of *Anthurium* is frequently used. Also, adding of BAP and 2-iP to the callus medium has been evaluated by different researchers. The concentrations of 2,4-D used in the callus medium is ranging from 0.08 mg/l to 1 mg/l 2,4-D.

Micropropagated plants require a developed root system to resist the external environmental conditions. Rooting of the shoots take place *in vitro*. Therefore, determination of the appropriate auxin type and levels in the media required to promote

Activated charcoal [AC] is added to medium for promoting root growth [11, 13, 15, 19, 20]. AC is composed of carbon and it is often used in plant tissue culture to absorb gases and dissolved solids. It is not a growth regulator but it has an ability to modify medium

There are several advantageous uses of charcoal on the type of culture. These are adsorption of secreted compounds from cultured tissues, decrements in the phenolic oxidations, pH changes of the medium to optimize for morphogenesis, prevention of unwanted callus growth, simulation of soil conditions because of the ability to promote root formation, capability to use in production of secondary plant products in culture

regeneration and NWT-3 media contains 750 mg/l NH4NO3.

The BA concentrations are changing between 0.1 mg/l and 1 mg/l.

**Figure 1.** *In vitro* propagation of *Anthurium* cultivars [Arizona]. The shoots with root were growth inplant tissue culture medium with AC [20].

## **3. The importance of subculturing in micropropagation**

In plant propagation applications, subculturing has an importance to prolong the life of plants and expand the number of cultured seedlings. At *in vitro* propagation of *Anthurium andreanum* cultivars, the number of shoots per explants was increased subsequent subcultures. Atak and Çelik [2009] observed that shoot multiplication for two *Anthurium andreanum* cultivars Arizona and Sumi was increased in the next multiplication stage. At every subculture, shoot numbers regenerated form nodal explants gradually increased [Table 3]. Bejoy et al.[2008] reported that multiplication was enhanced in the next multiplication stage. They succeeded to increase the rate of shoot production in the second multiplication stage.


Micropropagation of *Anthurium spp.* 251

**5. Discussion and conclusion** 

micropropagation methods should be determined.

*Istanbul Kultur University, Faculty of Science and Letters,* 

Cell. Dev. Biol-Plant 2003; 39 500-504.

*Department of Molecular Biology and Genetics, Ataköy, Istanbul, Turkey* 

ornamental pot plant.

conditions.

**Author details** 

**6. References** 

560.

2010; 6(8) 927-931.

Çimen Atak and Özge Çelik

In micropropagation studies, the success of the protocols depends on the variety of *Anthurium*, explant type, the components of the media used for shoot and root regenerations. Different combinations of plant growth regulators and additives used in relation to increase the regeneration potential of the explants should be evaluated for each cultivars to determine the efficient tissue culture protocol. In this chapter, we compared the explant types and tissue culture components for *Anthurium* species which is an important

Stages of the leaves show different response to propagation by indirect organogenesis. Explants prepared from brown leaves have higher callus formation rates in a shorter time than green leaf explants. Therefore, selection and using the right leaf explants at the appropriate leaf stage is the first step of establishing a successful tissue culture. Using different combinations of plant growth regulators and nitrogen additives should be evaluated to control the organogenesis for *Anthurium* varieties. NO3- : NH4+ balance in the growth medium has to be adjusted for each *Anthurium* varieties to obtain desired differentiation and growth. Developing an ideal acclimatization condition is important to increase the survival rate of micropropagated and rooted seedlings to adapt to *ex vivo*

In conclusion, the primary point to be remembered is the effects of genotypical differences on culture efficiencies. Different genotypes of varieties show different organogenesis responses in explant cultures. Therefore for each *Anthurium* varieties suitable

[1] Martin KP, Joseph D, Madassery J, Philip VJ. Direct shoot regeneration from lamina explants of two commercial cut flower cultivars of *Anthurium andreanum* Hort. *In Vitro*

[2] Rout GR, Mohapatra A, Jain SM. Tissue culture of ornamental pot plant: A critical review on present scenario and future prospects. Biotechnology Advances 2006; 24 531-

[3] Harb EM, Talaat NB, Weheeda BM, El-Shamy M, Omira GA. Micropropagation of *Anthurium andreanum* from shoot tip explants. Journal of Applied Sciences Research

Data presented as means with different letters within a column indicating significant differences at P<0.05 according to Duncan's Multiple range test. Each mean represented 5 replications.

Abbreviations: **Va1** =initial shoot [Shoot regeneration from callus cultures of Arizona variety] **Va2, Va3, Va4,**=

subcultures of Arizona variety, **Vs1** = initial shoot [Shoot regeneration from callus cultures of Sumi variety], **Vs2, Vs3, Vs4**= subcultures of Sumi variety.

**Table 3.** Shoot multiplication of *Anthurium andreanum* cultivars [20].

## **4. Acclimatization**

In micropropagation studies, the last and the critical step is acclimatization of the rooted seedlings to the environment. In this stage, plant losses have been due to different reasons [37,38]. Directly rooted shoots in soil show higher survival rate in the field than rooted under *in vitro* conditions. Therefore, there are several methods to high the survival rate of *in vitro* rooted shoots.

Cultured plants must adapt to low humidity, high light intensities and large temperature fluctuations with *ex vitro* acclimatization techniques. However these methods are expensive, time consuming and labor-intensive, *in vitro* acclimatization techniques have been improved. Using growth chambers which have relative humidity, controlled ventilation and possibility to change the components of the media make it possible to reduce the steps need for the process [37,39].

The success of acclimatization of *in vitro* cultured plants depends on the nutrients reserved in the leaves during development [40]. The important point in acclimatization is to keep the rooted plants in incubator in order to keep the humidity high.

Different acclimatization protocols for *in vitro* regenerated *A*.*andreanum* plantlets have been reported. Soilrite-perlite with the rate of 10:1, vermicompost and sand mixture [1:3], vermiculite and perlite [1:1], soil and organic humus [1:1] are the most used acclimatization mediums with the high survival ratios ranging from 60% to 98% [1,10,13].

During *in vitro* development stage, the cultural conditions such as humidity, air turbulence, CO2 concentration, sugar content in medium effect acclimatization ability of plants to *ex vivo* conditions. Therefore, for each *Anthurium* varieties, efficient acclimatization protocols have to be improved to prolong the success of micropropagation.

## **5. Discussion and conclusion**

250 Plant Science

Number of Explants

Duncan's Multiple range test. Each mean represented 5 replications.

**Table 3.** Shoot multiplication of *Anthurium andreanum* cultivars [20].

rooted plants in incubator in order to keep the humidity high.

to be improved to prolong the success of micropropagation.

mediums with the high survival ratios ranging from 60% to 98% [1,10,13].

**Vs4**= subcultures of Sumi variety.

**4. Acclimatization** 

*vitro* rooted shoots.

for the process [37,39].

Subculture **Arizona** Subculture **Sumi** 

Abbreviations: **Va1** =initial shoot [Shoot regeneration from callus cultures of Arizona variety] **Va2, Va3, Va4,**= subcultures of Arizona variety, **Vs1** = initial shoot [Shoot regeneration from callus cultures of Sumi variety], **Vs2, Vs3,** 

**Va1** 50 15. 64±1.69a **Vs1** 50 12.24±1.18a **Va2** 50 22.70±1.46b **Vs2** 50 15.98±1.36b **Va3** 50 26.76±1.30c **Vs3** 50 21.82±1.87c **Va4** 50 33.70±1.09d **Vs4** 50 26.96±1.46d Data presented as means with different letters within a column indicating significant differences at P<0.05 according to

In micropropagation studies, the last and the critical step is acclimatization of the rooted seedlings to the environment. In this stage, plant losses have been due to different reasons [37,38]. Directly rooted shoots in soil show higher survival rate in the field than rooted under *in vitro* conditions. Therefore, there are several methods to high the survival rate of *in* 

Cultured plants must adapt to low humidity, high light intensities and large temperature fluctuations with *ex vitro* acclimatization techniques. However these methods are expensive, time consuming and labor-intensive, *in vitro* acclimatization techniques have been improved. Using growth chambers which have relative humidity, controlled ventilation and possibility to change the components of the media make it possible to reduce the steps need

The success of acclimatization of *in vitro* cultured plants depends on the nutrients reserved in the leaves during development [40]. The important point in acclimatization is to keep the

Different acclimatization protocols for *in vitro* regenerated *A*.*andreanum* plantlets have been reported. Soilrite-perlite with the rate of 10:1, vermicompost and sand mixture [1:3], vermiculite and perlite [1:1], soil and organic humus [1:1] are the most used acclimatization

During *in vitro* development stage, the cultural conditions such as humidity, air turbulence, CO2 concentration, sugar content in medium effect acclimatization ability of plants to *ex vivo* conditions. Therefore, for each *Anthurium* varieties, efficient acclimatization protocols have

Number of Explants

Number of Shoots per Explant [Mean±SE]

Number of Shoots per Explant [Mean±SE] In micropropagation studies, the success of the protocols depends on the variety of *Anthurium*, explant type, the components of the media used for shoot and root regenerations. Different combinations of plant growth regulators and additives used in relation to increase the regeneration potential of the explants should be evaluated for each cultivars to determine the efficient tissue culture protocol. In this chapter, we compared the explant types and tissue culture components for *Anthurium* species which is an important ornamental pot plant.

Stages of the leaves show different response to propagation by indirect organogenesis. Explants prepared from brown leaves have higher callus formation rates in a shorter time than green leaf explants. Therefore, selection and using the right leaf explants at the appropriate leaf stage is the first step of establishing a successful tissue culture. Using different combinations of plant growth regulators and nitrogen additives should be evaluated to control the organogenesis for *Anthurium* varieties. NO3- : NH4+ balance in the growth medium has to be adjusted for each *Anthurium* varieties to obtain desired differentiation and growth. Developing an ideal acclimatization condition is important to increase the survival rate of micropropagated and rooted seedlings to adapt to *ex vivo* conditions.

In conclusion, the primary point to be remembered is the effects of genotypical differences on culture efficiencies. Different genotypes of varieties show different organogenesis responses in explant cultures. Therefore for each *Anthurium* varieties suitable micropropagation methods should be determined.

## **Author details**

Çimen Atak and Özge Çelik *Istanbul Kultur University, Faculty of Science and Letters, Department of Molecular Biology and Genetics, Ataköy, Istanbul, Turkey* 

### **6. References**


[4] George EF, Hall MA, Klerk JD. Plant propagation by tissue culture, Volume 1. The 9- Background, Springer, 2008.

Micropropagation of *Anthurium spp.* 253

[21] Bejoy M, Sumitha VR, Anish NP. Foliar Regeneration in *Anthurium andreanum* Hort. cv.

[22] Winarto B, Rachmawati F, Silva JAT. New basal media for half –anther culture of *Anthurium andreanum* Linden ex Andre cv. Tropical. Plant Growth Regulation 2011;65

[23] Jahan MT, Islam MR, Khan R, Mamun ANK, Ahmed G, Hakim, L. *In vitro* clonal propagation of *Anthurium* [*Anthurium andreanum* L.] using callus culture. Plant Tissue

[24] Chu CC, Wang CC, Sun CS, Hsu C, Yin KC, Chu CY, Bi FY. Establishment of an efficient medium for anther culture of rice through comparative experiments on the

[25] Gamborg O, Miller R, Ojimo K. Nutrient requirement suspensions cultures of soybean

[26] Murashige T, Skoog F. A revised medium fo rapid growth and bio-assays with tobacco

[27] Murashige T, Tucker DPH. Growth factor requirements of Citrus tissue culture. Proc. 1st

[29] Bajguz A, Piotrowska A. Conjugates of auxin and cytokinin. Phytochemistry 2009;70(8)

[30] Thomas TD. The role of activated charcoal in plant tissue culture. Biotechnology

[31] Asaduzzaman M, Asao T. Autotoxicity in beans and their allelochemicals. Scientia

[32] Kuehnle AR, Sugii N. Callus induction and plantlet regeneration in tissue culture of

[33] Teng WL. Regeneration of *Anthurium* adventitious shoots using liquid or raft culture.

[34] Chen FC, Kuehnle A, Sugii N. *Anthurium* roots for micropropagation and *Agrobacterium tumefaciens*-mediated gene transfer. Plant Cell, Tissue and Organ Culture 1997;49 71-74. [35] Joseph M, Martin KP, Mundassery J, Philip VJ. *In vitro* propagation of three commercial cut flower cultivars of *Anthurium andreanum*. Hortic. Indian Journal of Experimental

[36] Zhao Q, Jing J, Wang G, Wang JH, Feng YY, Xing HW, Guan CF. Optimization in Agrobacterium-medium transformation of *Anthurium andreanum* using GFP as a

[37] Diaz LP, Namur JJ, Bollati SA, Arce OEA. Acclimatization of Phalaeropsis and Cattleya

[38] Hazarika BN. Acclimatization of tissue cultured plants. Current Science 2003;85 1704-

[39] Lavanya M Venkateshwarlu B, Devi BP. Acclimatization of neem microshoots adaptable to semi-sterile conditions. Indian Journal of Biotechnology 2009;8 218-222.

obtained by micropropagation. Rev. Colomb. Biotechnol. 2010;12(2) 27-40.

[28] Nitsch JP and Nitsch C. Haploids plants from Pgrains. Science 1969;163 85-87.

Agnihothri. Biotechnology 2008;7(1)134-138.

nitrogen sources. Science Sinica 1975;18 659-668.

root cells. Experimental Cell Research 1968;50(1) 151-158.

tissue cultures. Physiologica Plantarum 1962;15 473-497.

Hawaiian *Anthuriums*. Hort Science 1991;26 919-921.

Plant Cell,Tissue and Organ Culture 1997;49 153-158.

reporter. Electronic Journal of Biotechnology 2010;13(5)1-11.

513-529.

957-969.

Cult. Biotech. 2009;19 61-69.

In. Citrus Symp. 1969;3 1155-1161.

Advences 2008;26 618-631.

Biology 2003;41 154-159.

1712.

Horticulturae 2012;134(1) 26-31.


[21] Bejoy M, Sumitha VR, Anish NP. Foliar Regeneration in *Anthurium andreanum* Hort. cv. Agnihothri. Biotechnology 2008;7(1)134-138.

252 Plant Science

Background, Springer, 2008.

Advances 2010; 28 462-488.

105 269-282.

2008;18(2) 113-122.

[4] George EF, Hall MA, Klerk JD. Plant propagation by tissue culture, Volume 1. The 9-

[5] Dobranszki J, Silva JAT. Micropropagation of apple – A review. Biotechnology

[6] Hamidah M, Karim AGA, Debergh P. Somatic embryogenesis and plant regeneration in *Anthurium scherzerianum*. Plant Cell, Tissue and Organ Culture 1997; 48 189-193. [7] Vargas TE, Mejias A, Oropeza M, Garcia E. Plant regeneration of *Anthurium andreanum*

[8] Dufour L, Guerin V. Growth, developmental features and flower production of *Anthurium andreanum* Lind. in tropical condition. Scientia Horticulturae 2003; 98 25-35. [9] Dufour L, Guerin V. Nutrient solution effects on the development and yield of *Anthurium adreanum* Lind. in tropical soilless conditions. Scientia Horticulturae 2005;

[10] Viegas J, Rosa da Rocha MT, Ferreira-Moura I, Lairia da Rosa D, Almeida de Souza J, Correa MGS, Telxelra da Silva JA. *Anthurium andraeanum* [Linden ex Andre] culture: *in* 

[11] Gantait S, Mandal N, Bhattacharyya S, Das PK. *In vitro* Mass Multiplication with pure genetic identity in *Anthurium andreanum* Lind. Plant Tissue Cult. Biotechnology

[12] Gantait S, Sinniah UR. Morphology, flow cytometry and molecular assessment of exvitro grown micropropagated anthurium in comparison with seed germinated plants.

[13] Gantait S, Mandal N. Tissue culture of Anthurium andreanum: Asignificant review and

[14] Silva JAT, Nagae S, Tanaka M. Effect of physical factors on micropropagation of

[15] Puchooa D. *In vitro* mutation breeding of Anthurium by gamma radation. International

[16] Lima FC, Ulisses C, Camara TR, Cavalcante UMT, Albuquerque CC, Willadino L. *Anthurium andraeanum* Lindl. cv. Eidibel *in vitro* rooting and acclimation with

[17] Yu Y, Liu L, Liu J, Wang J. Plant regeneration by callus-mediated protocorm-like body

[18] Maira 0, Alexander M, Vargas TE. Micropropagation and organogenesis of Anthurium andraeanum Lind cv. Rubun. Jain SM, Ochatt SJ.[Eds.] Protocols for *in vitro* propagation

[19] Nhut DT, Duy N, Vy NNH, Khue CD, Khiem DV, Vinh DN. Impact of *Anthurium* spp. genotype on callus induction derived from leaf explants and shoot and root regeneration capacity from callus. Journal of Applied Horticulture 2006;8(2) 135-137. [20] Atak C, Celik O. Micropropagation of *Anthurium andreanum* from leaf explants.

cv Rubrun. Electronic Journal of Biotechnology 2004;7(3) 285-289.

*vitro* and ex vitro. Floricult. Ornamental Biotechnology 2007;1 61-65.

future prospective. İnternational Journal of Botany 2010;6(3) 207-219.

arbuscular mycorrhizal fungi. Rev. Bras. Cienc. Arar. Recife 2006;1 13-16.

induction of *Anthurium andreanum.* Hod. Agric Sci. China 2009; 8 572-577.

of ornimental plants, Methods in Molecular Biology 2009;589 3-14.

African Journal of Biotechnology 2011;10(64) 13991-13998.

*Anthurium andreanum*. Plant Tissue Culture 2005;15(1) 1-6.

Journal of Agricultural Biology 2005;7 11-20.

Pakistan Journal of Botany 2009;41 1155-1161.


[40] Premkumar A, Mercado JA, Quesada MA. Effects of *in vitro* tissue culture conditions and acclimatization on the contents of Rubisco, leaf soluble proteins, photosynthetic pigments and C/N ratio. Journal of Plant Physiology 2001;158 835-840.

**Chapter 11** 

© 2012 Ahmed et al., licensee InTech. This is an open access chapter distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2012 Ahmed et al., licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

set up worldwide, especially in the developing countries due to cheap labour costs.

*In vitro* **Regeneration, Acclimatization and** 

A. Bakrudeen Ali Ahmed, S. Mohajer, E.M. Elnaiem and R.M. Taha

Tissue culture has been applied to diverse research techniques such as viral elimination, clonal propagation, gene conservation, *in vitro* fertilization, mutation, induction for genetic diversity, genetic transformation, protoplast isolation and somatic hybridization, secondary metabolite production and other related techniques. The commercial production of ornamental plants is growing worldwide. Its monetary value has significantly increased over the last two decades and there is a great potential for continued further growth in both domestic and international markets. About 156 ornamental genera are propagated through tissue culture in different commercial laboratories worldwide. About 212.5 million plants including 157 million ornamental plants amounting to 78% of the total production were reported [1]. These plants are over exploited due to their high medicinal value and hence, propagation of the plants by tissue culture may be mandatory, which offers a greater potential to deliver large quantities of disease-free, true-to-type healthy stock within a short span of time. Biotechnological interventions for *in vitro* regeneration, mass micropropagation and gene transfer methods in forest tree species have been practiced with success, especially in the last decade. Against the background of the limitations of long juvenile phases and lifespan, developments of plant regeneration protocols of ornamental species are gaining importance. Ornamental industry has applied immensely *in vitro* propagation approach for large-scale plant multiplication of elite superior varieties. During *in vitro* condition, plantlets are grown under fixed and controlled environment in sterile formulated medium which contained macronutrients, micronutrients, vitamins and plant growth regulators. After the plantlets reached optimum growth in the culture containers after a certain growth period, it can be transferred to *ex vitro* condition to allow continuous growth of the plantlets. As a result, hundreds of plant tissue culture laboratories have been

**Antimicrobial Studies of Selected** 

Additional information is available at the end of the chapter

**Ornamental Plants** 

http://dx.doi.org/10.5772/50690

**1. Introduction** 
