**2.3.2 Pathogen related protein (PR) and transcription factors:**

86 Soybean – Genetics and Novel Techniques for Yield Enhancement

Fig. 5. A) Fold change in expression of genes encoding allene oxide synthase (AOS); (B) fold change in expression of genes encoding allene oxide cyclase (AOC) in sysncytia of an incompatible reaction of SCN with soybean; and C) fold change in expression of genes encoding 12-Oxyphytodienoate reductase in syncytia in a compatible interaction (C) at 2, 5 and 10 dai (data from Ithal. *et al.* 2007b) and incompatible interaction (I) at 3, 6 and 9 dai

(data from Klink *et al.* 2009b). Genes are represented by GenBank numbers.

Pathogen related (PR) proteins are induced systemically by the interaction of a pathogen with its host (Van Loon & Van Strien, 1999). PR-1 and PR-2 are induced by SA (Ohishima *et al.,* 1990, Hennig *et al*., 1993), while basic PR genes are induced by JA (Niki *et al.* 1998). Genes encoding enzymes involved in JA synthesis were discussed above. Unfortunately, genes important to salicylic acid biosynthesis were either not represented on the microarray chip or were not annotated. However, genes encoding proteins of the PR-1, PR-2 and PR-5 families were up-regulated at 3, 6 and 9 dai in the incompatible interaction of soybean with SCN, suggesting that salicylic acid or its derivatives may be synthesized at these time points.

The PR1 gene, represented by CF806816, was increased 900, 2100 and 1600-fold at 3, 6, and 9 dai, respectively in the incompatible interaction of SCN with soybean, while the PR1 gene, represented by BQ628525, was over expressed 70, 240, 160-fold at 3, 6, and 9 dai (Klink *et al.*  2009b). During the compatible interaction, few PR1 genes were increased in expression and only one gene, represented by BU548404, was increased over 10-fold (Ithal. *et al.,* 2007b) and this was at 2 dai, when the nematode first initiates feeding. At 5 dai only two genes were increased in expression and this was at 5.6-fold and 2.8-fold, respectively. Only one PR-1 gene was increased in expression at 10 dai in the compatible interaction and that was only 5 fold increased in expression. Transcript levels of genes encoding PAL are also more strongly up-regulated in tomato roots displaying an incompatible interaction with the potato cyst nematode (*Globodera rostochiensis*), than in the compatible interaction (Uehara *et al.,* 2010). Arabidopsis roots infected with beet-cyst nematode (*Heterodera schachtii*), transcript levels of PR-1, PR-2, and PR-5 were increased, while PR-3 and PR-4 remained at similar levels to control plants (Hamamouch *et al.* 2010). Transcript levels of genes encoding PR-1 and PR5 were also increased in the incompatible interaction of Arabidopsis with the RKN, *M. incognita*, while transcript levels of PR-3 were elevated to a lesser extent. PR-3 and PR-4 are different types of chitinase. Seven chitinase genes are increased in expression at 3 dai in the incompatible reaction of soybean with SCN; three are approximately 20-fold over-expressed. At six dai, three genes encoding chitinase are expressed; one is 74-fold; A second gene is 33 fold increased in expression. No genes encoding chitinase are over-expressed in the incompatible reaction at 2 dai, and only one gene is over expressed at 5 and 10 dai, 6- and 15-fold, respectively (Fig 6). PR10 genes, represented by X60043, CF921432 and CF805736, are increased in expression 200-fold or more at all time points in both the compatible and incompatible interactions of SCN with soybean roots.

During the interaction of soybean roots with RKN, many genes encoding several PR proteinas were altered in expression (Ibrahim *et al.*, 2011). Transcripts of the gene encoding PR-1 were increased 78-fold at 12 dai in the compatible interaction of soybean roots with RKN. After prolonged infection by RKN at 10 wai, transcript levels of two genes encoding PR-1 were 17- and 350-fold increased. Genes encoding chitinase (PR-3 and PR-4) were down-regulated 4.6-fold at 12 dai in the compatible interaction of soybean roots with RKN, however, by 10 wai transcripts of two chitinase genes were up-regulated 15- to 26-fold, respectively. Transcripts of genes encoding PR-10 (SAM22) were increased 5- to 10-fold at 12 dai and remained at a similar level at 10 wai.

The increase in PR-1 protein suggests that there may be an increase in the level of salicylic acid. Interestingly, there are two different possible routes to salicylic acid production (Chen *et al*. 2009). Salicylic acid is known as a signal molecule for defense against nematodes (Branch *et al.,* 2004).

Changes in the Expression of Genes in Soybean Roots Infected by Nematodes 89

Fig. 7. Expression of genes encoding enzymes involved in phenylpropanoid biosynthesis (A) at 10 dai in a compatible reaction between Williams 82 with SCN and (B) at 6 dai in an incompatible reaction between cv. Peking and SCN are displayed on the KEGG pathway diagram. Enzyme commission numbers in the rectangles are provided by KEGG. Rectangles are colored light green for genes up-regulated in the first 50%, medium green for genes upregulated in the 50 to 75 quartile and dark green for genes up-regulated in the top 25 %s. Enzymes colored in red are encoded by down-regulated genes using a similar scheme. Enzymes colored in yellow are encoded by more than one gene and different copies of that gene are up- and down-regulated, respectively. Rectangles colored light gray indicate that the genes encoding those enzymes are not annotated in our soybean microarray database. Phenylalanine ammonia-lyase (EC 4.3.1.34; PAL) can be considered a control point for entry into the phenylpropanoid pathway. There is no major change in expression of genes encoding PAL in the compatible interaction, however at 3, 6 and 9 dai in the incompatible interaction genes encoding PAL are increased in expression, thus suggesting an increased metabolic flow into the pathway. Genes encoding PAL and represented by BI701520, CK606172, and AW351172 are 20- to more than 40-fold increase in expression over that time course (Klink *et al.* 2007a). Increased PAL enzyme activity has been noted in resistant tomato roots infected with RKN, while PAL activity was depressed in susceptible tomato roots (Brueske, 1980). Similarly, in potato PAL activity is higher in resistant plants (Giebel, 1973) . Certainly certain genes involved in isoflavonoid production are increased in expression in the incompatible reaction. For example, the gene encoding chalcone synthase (EC 2.3.1.74),represented by BQ081473, is more than 40-fold increased in expression at 3 and 9 dai in the incompatible interaction, but there is no change in the compatible interaction, while one gene encoding chalcone isomerase is eleveated 4-, 6- and 17-fold in the

incompatible interaction (Klink *et al.* 2007b).

Fig. 6. Fold increase in expression of chitinase genes in the compatible (C) interaction (data from Ithal. *et al.* 2007b) and incompatible interaction (I) at 3, 6 and 9 dai (data from Klink *et al.* 2009b). Genes are represented by GenBank numbers.

The pathway that has the most scientific support involves isochorismate synthase (Wildermuth *et al*. 2001) and is not represented or is not annotated on the microarray. The other pathway involves phenylalanine. In the latter pathway, we found a high increase in the tyrosine aminotransferase enzyme (EC:2.6.1.5) which would lead to high level of phenylalanine. Genes encoding phenylalanine ammonia-lyase (EC:4.3.1.24) and salicylate 1 monooxygenase (1.14.13.-) were over-expressed at 6.9 and 2.9 F.C, respectively. Loon *et al.* (2006) reported that the PR-1- type proteins and also proteinase inhibitors were induced in abscission zones, which suggest the involvement of these proteins in cell wall loosening and degradation of the scarified cells as a defense response against fungal and bacterial pathogens. Also, transgenic tobacco over-expressing PR-1 was more resistant to blue mold and black shank caused by *Peronospora tabacina* and *Phytophthora parasitica f. sp. nicotianae*, respectively (Loon *et al.,* 2006). In addition, PR-3 and PR-4 showed chitinase activity that is required for embryogenesis during the globular stage in carrot (Loon *et al.,* 2006). Genes encoding PR-3 and PR-4 family proteins are reported to be up-regulated by jasmonic acid and ethylene (Niki *et al.,* 1998). Also, PR-4 showed ribonuclease activity against fungal protein in wheat (Loon *et al*., 2006).

### **2.3.3 Phenylpropanoid biosynthesis**

The phenylpropanoid pathways leads to the synthesis of coumarins, flavonoids, phytoalexins, lignins, and lignans, all which can play roles in plant defense. Several genes encoding enzymes in this complex pathway are up regulated in the incompatible interaction at 6 dai (Fig . 7; data from Klink et al. 2007b). And there are notable differences in the expression of genes encoding enzymes in this pathway between the compatible interaction at 2, 5 and 10 dai and the incompatible reaction at 6 and 9 dai. One major difference is in the expression of the genes encoding enzymes involved in the production of phenylpropanoids.

Fig. 6. Fold increase in expression of chitinase genes in the compatible (C) interaction (data from Ithal. *et al.* 2007b) and incompatible interaction (I) at 3, 6 and 9 dai (data from Klink *et* 

The pathway that has the most scientific support involves isochorismate synthase (Wildermuth *et al*. 2001) and is not represented or is not annotated on the microarray. The other pathway involves phenylalanine. In the latter pathway, we found a high increase in the tyrosine aminotransferase enzyme (EC:2.6.1.5) which would lead to high level of phenylalanine. Genes encoding phenylalanine ammonia-lyase (EC:4.3.1.24) and salicylate 1 monooxygenase (1.14.13.-) were over-expressed at 6.9 and 2.9 F.C, respectively. Loon *et al.* (2006) reported that the PR-1- type proteins and also proteinase inhibitors were induced in abscission zones, which suggest the involvement of these proteins in cell wall loosening and degradation of the scarified cells as a defense response against fungal and bacterial pathogens. Also, transgenic tobacco over-expressing PR-1 was more resistant to blue mold and black shank caused by *Peronospora tabacina* and *Phytophthora parasitica f. sp. nicotianae*, respectively (Loon *et al.,* 2006). In addition, PR-3 and PR-4 showed chitinase activity that is required for embryogenesis during the globular stage in carrot (Loon *et al.,* 2006). Genes encoding PR-3 and PR-4 family proteins are reported to be up-regulated by jasmonic acid and ethylene (Niki *et al.,* 1998). Also, PR-4 showed ribonuclease activity against fungal

The phenylpropanoid pathways leads to the synthesis of coumarins, flavonoids, phytoalexins, lignins, and lignans, all which can play roles in plant defense. Several genes encoding enzymes in this complex pathway are up regulated in the incompatible interaction at 6 dai (Fig . 7; data from Klink et al. 2007b). And there are notable differences in the expression of genes encoding enzymes in this pathway between the compatible interaction at 2, 5 and 10 dai and the incompatible reaction at 6 and 9 dai. One major difference is in the expression of the genes encoding enzymes involved in the production of phenylpropanoids.

*al.* 2009b). Genes are represented by GenBank numbers.

protein in wheat (Loon *et al*., 2006).

**2.3.3 Phenylpropanoid biosynthesis** 

Fig. 7. Expression of genes encoding enzymes involved in phenylpropanoid biosynthesis (A) at 10 dai in a compatible reaction between Williams 82 with SCN and (B) at 6 dai in an incompatible reaction between cv. Peking and SCN are displayed on the KEGG pathway diagram. Enzyme commission numbers in the rectangles are provided by KEGG. Rectangles are colored light green for genes up-regulated in the first 50%, medium green for genes upregulated in the 50 to 75 quartile and dark green for genes up-regulated in the top 25 %s. Enzymes colored in red are encoded by down-regulated genes using a similar scheme. Enzymes colored in yellow are encoded by more than one gene and different copies of that gene are up- and down-regulated, respectively. Rectangles colored light gray indicate that the genes encoding those enzymes are not annotated in our soybean microarray database.

Phenylalanine ammonia-lyase (EC 4.3.1.34; PAL) can be considered a control point for entry into the phenylpropanoid pathway. There is no major change in expression of genes encoding PAL in the compatible interaction, however at 3, 6 and 9 dai in the incompatible interaction genes encoding PAL are increased in expression, thus suggesting an increased metabolic flow into the pathway. Genes encoding PAL and represented by BI701520, CK606172, and AW351172 are 20- to more than 40-fold increase in expression over that time course (Klink *et al.* 2007a). Increased PAL enzyme activity has been noted in resistant tomato roots infected with RKN, while PAL activity was depressed in susceptible tomato roots (Brueske, 1980). Similarly, in potato PAL activity is higher in resistant plants (Giebel, 1973) . Certainly certain genes involved in isoflavonoid production are increased in expression in the incompatible reaction. For example, the gene encoding chalcone synthase (EC 2.3.1.74),represented by BQ081473, is more than 40-fold increased in expression at 3 and 9 dai in the incompatible interaction, but there is no change in the compatible interaction, while one gene encoding chalcone isomerase is eleveated 4-, 6- and 17-fold in the incompatible interaction (Klink *et al.* 2007b).

Changes in the Expression of Genes in Soybean Roots Infected by Nematodes 91

reaction. In general, genes encoding pathogenesis-related proteins are more highly expressed in the incompatible interaction and a gene encoding phenylalanine ammonia lyase is much more highly expressed in the incompatible interaction of soybean roots with SCN. Phenylalanine ammonia lyase is major gateway to phenylpropanoid metabolism and to the synthesis of numerous secondary compounds involved in plant defense, All of these data indicate that there is a stronger production of transcripts of genes encoding proteins involved in the plant defense response in the incompatible interaction, while transcripts of many of these genes are lower or the genes are down-regulated leading to a weaker defense response in the compatible reaction of soybean roots to SCN. Gene expression studies performed in soybean has resulted in the understanding gene expression during infection by SCN. The challenge to scientists now is in testing the function genes to understand the molecular circuitry occurring between plants and their parasitic nematodes so new methods

The authors gratefully acknowledge support from United Soybean Board project number Y9254, the US-Egypt Science and Technology project number BIO8-001-002 and from the BioGreen 21 Program (no. PJ007031), Rural Development Administration, Republic of Korea

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**4. Acknowledgments** 

**5. References** 

Fig. 8. Expression of genes encoding phenylalanine ammonia-lyase (EC 4.3.1.24) at 3, 6 and 9 dai after infection with SCN in an incompatible interaction with soybean roots (Data from Klink *et al.* 2007b) phenylalanine ammonia-lyase (PAL; EC 4.3.1.24; Fig. 7; Data from Klink *et al.* 2007b).

While microarray studies of genes expressed in the incompatible reaction of soybean plants against SCN revealed an increase in transcript levels of certain genes encoding enzymes involved in glycolysis/gluconeogenesis, jasmonic acid biosynthesis, phenylpropanoid biosynthesis, pathogenesis related proteins, flavonoid biosynthesis, and the methionine salvage pathway (Klink *et al.,* 2010; Alkharouf *et al*., 2006), the expression of many genes encoding proteins having regulatory and signaling functions, such as cyclins, phosphokinases and transcription factors, were also affected. Genes encoding enzymes belonging to pathways depicted in KEGG and that were highly preferentially expressed were related to those KEGG pathways using PAICE software (Hosseini *et al.,* in preparation) to make interpretation of the data easier. Thus, relationships among genes and pathways were recognized with less difficulty.
