**2. Characteristics of ALSV vector**

ALSV is a rare, naturally occurring plant virus isolated from an apple tree. ALSV has only been detected in a single apple tree, excluding experimentally infected plants. The infected tree was grown in the orchard of a Japanese fruit tree research institute (Morioka, Iwate, Japan). The cultivar of this apple is 'Indo', named after Indiana State in the United States of America. The Indo apple used to be popular in Japan, but has now been largely replaced with other cultivars such as Fuji and Orin. ALSV does not naturally spread to nearby apple trees in the orchard [8], but it can infect the seedlings of apple cultivars in the experiments [9–11]. Moreover, ALSV is a 'latent' virus. This means that apple plants (and fruits) infected by ALSV do not show any discernible changes, such as yellow spots, inferior growths or twisted leaves. Thus, ALSV does not induce disease, but such latent characteristics are advantageous for plant biotechnology. With the ALSV vector, we can specifically upregulate (activate) or downregulate (inactivate) functions of target genes, for example, among the 57,000 genes in the apple genome [12], with the ALSV vector. There remain other advantages of the ALSV vector. ALSV evenly infects plant tissues and then enables even silencing (inactivation) of apple genes [11, 13]. ALSV can also infect other plants and upregulate/downregulate specific genes. For instance, ALSV can infect to soybean, petunia, pea, cucumber, pear, rose, Eustoma and Japanese gentian. ALSV is latent in many plant species [14, 15]. Unlike genetic transformation, whose infection rates greatly differ between cultivars of the same crop, ALSV does not typically select cultivars. Exceptions are tomato and almond, where ALSV can infect only part of the cultivars.

traits. One generation time (from germination, flowering, seed set and germination of the next-generation seedling) is usually several months in herbaceous plants and several years in trees under field conditions [1]. Thus, crossing of herbaceous vegetables and ornamental flowers can be performed every year, while breeding of fruit trees may only be performed once every several years. If flowering could be accelerated in horticultural plants, we would be able to cross vegetables and flowers many times each year and cross fruit trees every year. In order to cross vegetables several times in a year, problems related to the seasonality and day-length sensitivity of plant flowering also need to be addressed. Plants are either shortday, long-day or day-neutral (an aspect of the photoperiodism, the response to day length). Short-day plants flower in autumn and long-day plants flower in spring. Day-neutral plants flower in any growing season. Day length is now controlled by artificial lighting, but biotechnology to accelerate flowering will also solve the problem of day-length sensitivity without the need for regulating day length using lighting equipment. Alternatively, daylength sensitivity is also one of the important traits of horticultural plants. Ornamental flowers are harvested only when the plants set flowers. Fruits can be harvested only after the flowering seasons. Because of stable responses of horticultural plants to day length, farmers make great efforts to control time of flowering. Such artificial controls are possible only with lighting equipment, but the majority of crops are produced using sunlight alone. Gene modification by biotechnology such that a series of cultivars with different daylength responses are prepared for major horticultural cultivars will thus benefit agricultural

Basic studies using model plants have revealed the genes controlling flowering time in higher plants. After a functional gene is isolated in model plants, that gene and its homologs are often found to exert the same function in other plant species. This means that the flowering of vegetables, ornamental flowers and fruit trees can be controlled by this gene [2]. The name of the gene controlling plant flowering is '*Flowering locus T*' (*FT*). Initially, plant biologists aimed to express the *FT* gene in crops by transformation, which actually accelerated flowering [3–7]. This suggested the functional use of *FT* in controlling flowering time, but transgenic plants are not typically applicable to crop production. Plant biologists then attempted nontransgenic expression of *FT* in crops. To our knowledge, there have been no scientific papers demonstrating the control of flowering time by application of FT protein, *FT* DNA or *FT* RNA without genetic transformations or spontaneous mutations in plant genomic DNA. We used a virus vector derived from *Apple latent spherical virus* (ALSV) to deriver *FT* RNA and express FT protein in plant tissues. This system turned out to be very successful in promoting flowering in horticultural plants. This chapter explains in detail the characteristics of the ALSV vector, activities of *FT* and its related genes, and how the ALSV vector is used to promote

ALSV is a rare, naturally occurring plant virus isolated from an apple tree. ALSV has only been detected in a single apple tree, excluding experimentally infected plants. The

production.

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flowering.

**2. Characteristics of ALSV vector**

The protocol for preparation and infection of the ALSV vector is precisely described in our recent publication [15]. Briefly, plasmid DNAs for expression of ALSV RNAs are prepared by ordinary procedures, introduced into *Agrobacterium*, and 'Agro-inoculated' into benthamiana tobacco (*Nicotiana benthamiana*), which is readily infected by plant viruses. Virus particles are extracted from infected leaves; viral RNAs are extracted and introduced into horticultural plants by 'particle bombardment'. The precise structures of ASLV and ALSV vector are explained below.

ALSV consists of two genomic RNAs: ALSV-RNA1 and ALSV-RNA2. **Figure 1** shows the whole sequences of ALSV-RNA1 and ALSV-RNA2. The lengths of ALSV-RNA1 and ALSV-RNA2 are 6812 bases and 3384 bases, respectively. Similar to genomic RNAs of other plant viruses [16], genomic RNAs of ALSV encode single polyproteins. Polyproteins are translated as fused proteins and are digested into individual units by protease. ALSV-RNA1 encodes enzymes such as protease, helicase and RNA polymerase. ALSV-RNA2 encodes a movement protein and capsid proteins. The cleavage sites of the polyprotein were confirmed for ALSV-RNA2 by peptide sequences, whereas the cleavage sites of the polyprotein encoded by ALSV-RNA1 were just deduced from the peptide sequence [17]. The movement protein and the three capsid proteins are all necessary for cell-to-cell movement of ALSV within plant tissues [18].

ALSV is expected to be formed in plants such as *N. benthamiana* if ALSV-RNA1 and ALSV-RNA2 sequences are expressed simultaneously. Expression should not necessarily be performed by genetic transformation: faster and easier protocols for transient expression are usually adopted. Once ALSV is formed in the cells of *N. benthamiana*, it will regenerate and spread autonomously. Three sets of different plasmid vectors have been developed for ALSV vectors (**Table 1**).


**Figure 1.** Nucleotide sequences of ALSV genomic RNAs. Protein units are shown with different colours, between the non-coding sequences at the heads and the tails of ALSV genomic RNAs. The following proteins are encoded in ALSV-RNA1 in this order: PRO-co (protease cofactor), HEL (NTP-binding helicase), Vpg (viral protein genome-linked), C-PRO (cysteine protease), and POL (RNA polymerase). The following proteins are encoded in ALSV-RNA2 in this order: MP (movement protein), capsid proteins: Vp25, Vp20, and Vp24. Genbank/INSDC accessions and nucleotide lengths are shown in parentheses.

ALSV Vector Substantially Shortens Generation Time of Horticultural Plants http://dx.doi.org/10.5772/intechopen.70317 73


From left column to right column are shown: vector names, expressed genes, plasmid backbone origins, antibiotics for plasmid selections, and reference papers.

PVY, *Potato virus Y*; ClYVV, *Clover yellow vein virus*.

**Table 1.** List of plasmid vectors.

**Figure 1.** Nucleotide sequences of ALSV genomic RNAs. Protein units are shown with different colours, between the non-coding sequences at the heads and the tails of ALSV genomic RNAs. The following proteins are encoded in ALSV-RNA1 in this order: PRO-co (protease cofactor), HEL (NTP-binding helicase), Vpg (viral protein genome-linked), C-PRO (cysteine protease), and POL (RNA polymerase). The following proteins are encoded in ALSV-RNA2 in this order: MP (movement protein), capsid proteins: Vp25, Vp20, and Vp24. Genbank/INSDC accessions and nucleotide

lengths are shown in parentheses.

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pEALSR1 and pEALSR2 were the original vectors used for ALSV preparation. High concentrations of these vectors are prepared from colon bacteria (*Escherichia coli*) culture and rub-inoculated (inoculated with SiC, carborundum) onto the leaves of quinoa (*Chenopodium quinoa*) plants. The so-called agro-inoculation method was more convenient, and then new vectors pBICAL1 and pBICAL2 were developed, which are typically used in our experiments. These vectors are prepared in *E. coli* and transformed into Agrobacterium (*Agrobacterium tumefaciens*). *A. tumefaciens* harbouring ALSV vectors are injected into the leaves of *N. benthamiana* for transient expression of ALSV-RNAs. pCALSR1 and pCALSR2 undergo the same procedures with pBICAL1 and pBICAL2, but appear to merit higher concentrations in *E. coli*. The infection rate of 'wild-type' ALSV without modification is almost 100% in *N. benthamiana*. ALSV vectors with exogenous insertion sequences in either cloning sites suffer from much lower infection rates in *N. benthamiana*. To compensate for the reduced infection rate, 'silencing suppressor' protein HC-Pro derived from other viruses (ClYVV or PVY, [22]) is also transiently expressed when ALSV-RNAs are agro-inoculated into *N. benthamiana*. In conclusion, triple agro-inoculation of a mixture of ALSV-RNA1 clone, ALSV-RNA2 clone and HC-Pro clone enables efficient formation of ALSV vectors.

It was not easy to insert exogenous nucleotide sequences into ALSV. After many trials (Li C et al., unpublished data), three cloning sites were successfully used for nucleotide insertions (**Figure 2**) [9, 20]. One cloning site is located immediately after the stop codon of the polyprotein encoded by ALSV-RNA1 (named the SM site, after restriction sites for *Sal*I and *Mlu*I; **Figure 2A**). Another cloning site is located at the middle of the polyprotein encoded by ALSV-RNA2, between MP and Vp25 (named the XSB site, after restriction sites for *Xho*I, *Sma*I and *Bam*HI; **Figure 2B**). Nucleotide sequences around the XSB site are complicated. First, nucleotide sequences around the protease-digested site (QG site) encoding 10 amino acid residues (LLEGQGPDFT) were duplicated. Second, sequences of the restriction sites were introduced

**Figure 2.** Structures of three cloning sites in ALSV vector. (A) The SM site near the tail of ALSV-RNA1. (B) The XSB site at the middle of the ALSV-RNA2. Introduced mutations are indicated with '+' letters. (C) The MN site near the tail of ALSV-RNA2. Introduced restriction sites are shown with grey letters in (A)–(C).

between the duplicated sequences. Finally, silent mutations (without amino acid changes) were introduced at the third nucleotides in each 10 codons surrounding the cloning site. These nucleotide modifications around the XSB site were necessary to insert exogenous nucleotide sequences at this site. The protease-digested site was duplicated such that both sites (before and after the inserted peptide) are cleaved by protease. Another special requirement for the virus vector is that the insertions are stably held by the virus. Nucleotide sequences in viruses, particularly repetitive sequences, are quite easily deleted (lost) probably via homologous recombination [23–25]. To mitigate such unfavourable reactions, mutations had been introduced around the XSB site without changing the protease-digested amino acid sequence. The third cloning site is located immediately after the stop codon of the polyprotein encoded by ALSV-RNA2 (named the MN site, after the restriction sites of *Mlu*I and *Nco*I; **Figure 2C**). These cloning sites were constructed by Li C, based on pEALSR vectors, and then copied to the other vectors.

Among the three cloning sites of the ALSV vector (the SM site, the XSB site and the MN site), genes can be expressed only by their insertion into the XSB site. At the XSB site, the inserted genetic sequence is translated as a part of polyprotein, followed by digestion with protease. Cleaved proteins are expected to be attached with short peptides at both the N-terminus and the C-terminus , which derive from the protease-digested sites and the cloning site. These small attached peptides do not seem to affect the activity of inserted protein in most cases, as is experienced for transgenes introduced into transgenic plants.

All three cloning sites can be used for VIGS (virus-induced gene silencing). VIGS is a viral counterpart for gene silencing driven by antisense or inverted-repeat sequences expressed by genetic transformation [26, 27]. The virus itself is a natural target of gene silencing, then a nucleotide sequence inserted at any of the cloning sites of ALSV are silenced (degraded) by silencing mechanisms equipped in plant cells. Endogenous mRNAs harbouring the same nucleotide sequences with the inserted sequence in ALSV vector are also degraded, resulting in gene silencing. Any nucleotide sequences can be inserted at the SM site or at the MN site in principle. The nucleotide sequences inserted at the XSB site must be 'in frame' with the polyprotein, and this means the length of the inserts is a multiple of 3 and that each 'codon' encodes an amino acid without any stop codons (TAA, TAG or TGA as DNA sequences). Sizes of the inserted sequences are determined by the balance between the expected degree of silencing and potential risk of deletions (of the inserts from ALSV). Longer inserts will cause stronger silencing, but they will be more easily deleted from the ALSV vector. A length of 200 bases (or 201 bases at the XSB site) appears to be near the optimal size, with strong silencing and a relatively low possibility of deletions. Inserts shorter than 200 bases may not cause strong silencing, but the actual degree of silencing and frequency of deletions vary, depending on the inserted nucleotide sequences [14]. Insertion of nucleotide sequences at the XSB site is empirically simpler than insertions at the SM site or at the MN site. Insertions at the MN site strongly reduce the rate of viral infection, and they are also easily deleted. Insertions at the SM site have somewhat milder effects on infection rates and deletions. Insertions at the XSB site do not strongly reduce infection rate, but strongly suppress gene expression.

Model studies of silencing an endogenous gene with viral vectors often target the *Phytoene desaturase* (*PDS*) gene. **Figure 3** shows an *N. benthamiana* plant whose *PDS* gene was silenced by an ALSV vector. PDS catalyses the synthesis of carotenoids. Plants silenced in *PDS* expression become white, because they cannot accumulate both carotenoids and chlorophylls. This phenomenon (photo-bleaching) is believed to be caused by rapid 'photo-oxidation' of chlorophylls in the absence of photo-protective carotenoids in plant cells [28]. The representative mechanism of photo-protection by carotenoid would be non-photochemical quenching (NPQ) catalysed by PsbS protein and xanthophyll cycle. As Arabidopsis and rice mutants deficient in NPQ do not develop such white leaves caused by *PDS* silencing [29, 30], another major and unidentified photo-protective mechanism will be exerted by carotenoids in plant cells.

between the duplicated sequences. Finally, silent mutations (without amino acid changes) were introduced at the third nucleotides in each 10 codons surrounding the cloning site. These nucleotide modifications around the XSB site were necessary to insert exogenous nucleotide sequences at this site. The protease-digested site was duplicated such that both sites (before and after the inserted peptide) are cleaved by protease. Another special requirement for the virus vector is that the insertions are stably held by the virus. Nucleotide sequences in viruses, particularly repetitive sequences, are quite easily deleted (lost) probably via homologous recombination

**Figure 2.** Structures of three cloning sites in ALSV vector. (A) The SM site near the tail of ALSV-RNA1. (B) The XSB site at the middle of the ALSV-RNA2. Introduced mutations are indicated with '+' letters. (C) The MN site near the tail of

ALSV-RNA2. Introduced restriction sites are shown with grey letters in (A)–(C).

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**Figure 3.** Silencing of *PDS* gene by ALSV vector. (A) An ALSV vector having insertion of a 201-base tobacco *PDS* gene fragment [ALSV-XSB(NtPDS-201)]. (B) The ALSV vector was infected into *N. benthamiana*. NI, non-inoculated ('healthy') plant.

Based on the degree of silencing of the apple *RubisCO small subunit* (*rbcS*) gene with the same 201-base insertion sequence, silencing effects of the three cloning sites differ from one another, and they are greater in the following order: the XSB site > the MN site > the SM site [31]. Again, insertion of a 201-base fragment of the target gene in the XSB site of the ALSV vector is recommended for efficient gene silencing with a lower risk of deletions. The SM site and the MN site are also available to silence additional target genes, by inserting different sequences at each cloning sites. Such simultaneous expression/suppression of different genes is possible, but such vectors typically show low infection rates. The MN site was also used for virus-induced transcriptional gene silencing (VITGS) in *N. benthamiana* and petunia, where upstream sequences (promoters) of the target genes are methylated by virus vectors [20]. The nucleotide sequence of an apple gene promoter was also mutated and inserted at the XSB site for infection to apple plants [15].
