**2.3.1 Alpha-linolenic acid and jasmonic acid biosynthesis**

84 Soybean – Genetics and Novel Techniques for Yield Enhancement

When a nematode invades a plant root, it must repress or control the plant defense response, so it can successfully establish its permanent feeding site (Caillaud *et al*., 2001). These defense responses may include the production of jasmonic acid and salicylic acid, the hypersensitive response, cell wall strengthening, the production of pathogenesis related (PR) proteins, and other cellular defense responses. There are changes in the expression of genes involved in many of these defense responses in both compatible and incompatible interactions of SCN with soybean and with RKN and soybean in the compatible interaction. Many of the same genes are altered in expression in both the compatible and incompatible interaction. However, the amount of change in transcript abundance may be very important and in some cases a gene is up regulated in one interaction and down regulated in another

Fig. 4. (A) Fold change in expression of genes encoding xyloglucanases in syncytia in a compatible interaction (S) at 2, 5 and 10 dai. Data from Ithal. *et al. (*2007b) and (B) an incompatible interaction at 3, 6 and 9 dai Data from Klink *et al*.(2009a). Genes are

represented by GenBank numbers.

**2.3 Plant defense system** 

interaction

The pathway leading to jasmonic acid biosynthesis is one of the pathways associated with pathogen resistance that was significantly affected by both SCN and RKN infection. In soybean there are several lipoxygenase gene family members. Several members of this gene family are expressed higher in the compatible reaction of SCN with soybean at 2, 5 and 10 dai, specifically CF808603, CD409280 and BM092012, which are elevated 2.4- to 6.3-fold (Data from Ithal *et al.* 2007b). In contrast, in the incompatible reaction of SCN with soybean, several members of the gene family are down-regulated, while others are up- regulated, ranging between approximately -22- to 22-fold (Klink *et al.* 2007a). Genes encoding allene oxide synthase (AOS) and allene oxide cyclase (AOC) are not greatly changed in the compatible interaction at 2, 5 and 10 dai fold (data from Ithal *et al.* 2007b). However, three members of the AOS gene family are down regulated in the incompatible interaction at 3 dai, while syncytia are forming. Then expression of one gene family member is increased at 6 and 9 dai as the syncytia collapse and become non-functional (Fig. 5 A; Klink *et al.* 2007b). Expression of genes encoding AOC is increased in syncytia during the incompatible reaction, especially at 3 dai, then decreases in expression at 6 and 9 dai (Fig. 5B; data from Klink *et al.* 2007a). A genes encoding 12-oxyphytodienoate reductase 1 (OPR1), represented by BI968944, is strongly down-regulated in the compatible interaction of SCN with soybean roots (Ithal *et al.* 2007b), while a gene encoding OPR3, represented by BU765938, is up regulated 14-fold at 6 dai in the incompatible reaction (Fig 5C; Klink *et al.* 2007b). Thus, there is an increase in transcripts for specific gene members encoding enzymes through the pathway leading to JA biosynthesis in the incompatible reaction of SCN with soybean roots, while there is either no effect on genes encoding AOS and AOC or a decrease in transcript levels in the case of the gene encoding OPR1 in the compatible reaction. JA biosynthesis is one of the pathways affected in soybean roots by infection with RKN at 12 dai and 10 wai (Ibrahim *et al.,* 2011). At 12 dai, most of the genes encoding enzymes encoding lipoxygenase family members were up-regulated. Lipoxygenase is important in the biosynthesis of oxylipins and it is important in the response of plants during wounding and attack by pathogens (Gobel *et al*, 2001). Reduction of the expression of the gene encoding this lipoxygenase resulted in an increase in susceptibility of transgenic potato plants to insect attack (Gobel *et al.*, 2001). Over-expression of the gene encoding lipoxygenase could mean a high accumulation of 9-HPOTrE, as it is one of the major products of lipoxygenase (Fig. 6). Interestingly, 9-HPOTrE is involved in the activation of the plant defense response directly or through its metabolites. In potato plants, 9-HPOTrE is produced in response to injury or infection. The role of 9-HPOTrE in the plant defense response suggests that there may be a new pathway leading to LOX-mediated defense responses (Reddy *et al.,* 2000). The same results have been observed in pigeon pea seedlings after infection with *Fusarium udum* (Reddy *et al.,* 2000).

Transcript abundance of genes encoding lipoxygenase was much lower at 10 wai (weeks after infection) than at 12 dai in roots infected by RKN (Ibrahim *et al.* 2011). Three of seven gene family members encoding lipoxygenase were down-regulated. Also, all of the allene oxide synthase gene family members were greatly down-regulated at 10 wai This suggests that at 12 dai the plant defense system is still struggling to fight the infection, but after prolonged infection (10 wai) most of the genes that encode enzymes responsible for the production of jasmonic acid were turned off in the compatible interaction. Genes in this pathway could be a target for testing to determine if resistance to nematode infection can be increased in transformed plants by over-expression of these genes.

Changes in the Expression of Genes in Soybean Roots Infected by Nematodes 87

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

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

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

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

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

incompatible interactions of SCN with soybean roots.

dai and remained at a similar level at 10 wai.

(Branch *et al.,* 2004).

points.

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.
