Gene expression analysis
For the gene expression studies, five defense related genes that were identified during the analysis of M. truncatula responses to herbivory by S. exigua caterpillar with either normal or impaired labial salivary secretions were focussed (Darwish et al., 2008). The genes that were found to be differentially expressed in response to caterpillar herbivory encoded a ribulose-1, 5-bisphosphate carboxylase/oxygenase activase (MtRCA), a strictosidine synthase-like protein (MtSTR), a ring zinc finger protein (MtRFP), an unknown protein (MtUNK) and a receptor-like protein kinase (MtRPK). Along with these genes, genes encoding hevein-like protein, MtHEL (PR4), and vegetative storage protein, (MtVSP), were included as marker genes of AP2/ERF branch and MYC2 branch of JA pathway respectively. Gene encoding pathogenesis related protein 1 (MtPR1), which is a marker of NPR1 depended SAR pathway, and an ethylene-responsive element binding protein (MtEBP), and were analyzed.
All the genes studied but MtEBP, MtVSP and MtPR1, were down-regulated due to caterpillar herbivory compared to control in wild type M. truncatula. The expression of gene encoding EBP was not affected due to caterpillar herbivory. Significant increase in the level of VSP transcript in caterpillar infested plants compared to control plants confirmed the induction of MYC2 branch of JA-mediated defense pathway in response to herbivory. However, the levels of HEL transcripts were significantly supressed due to herbivory. This could be due to the antagonistic interaction between the MYC2 branch and AP2/ERF branch of JA pathway. Similar effect was seen after herbivory by S. exigua in tobacco…………….(REF). In M. truncatula, no labial-salivary specific effect was observed in the expression of studied defense related genes except MtPR1. Gene encoding PR1 protein, which is an important marker of SAR pathway, was significantly induced in plants fed by caterpillars with intact spinneret but not by cauterized caterpillars. This can lead to the conclusion that effectors in caterpillar’s labial saliva stimulate an antagonistic pathway to suppress the plant induced JA pathway in favour of caterpillars.
When the above mentioned defense related genes were studied in an ethylene insensitive skl mutants, the expression of MtPP and MtRCA genes due to normal and cauterized caterpillar herbivory were down-regulated. Since these genes were supressed in wild type plants also after herbivory, this indicates that ethylene is not involved in the expression of these genes. Constitutive expression level of MtSTR was lower in skl mutant than wild type plants which might indicate that ethylene response is required for the expression of this gene. At the same time, feeding by both caterpillars, with or without intact spinneret, had no effect in the expression of MtSTR in skl mutant. Unlike in wild type M. truncatula, caterpillars with intact and ablated spinneret did not suppress the expression of two regulatory genes, MtRPK and MtRFP , in skl mutant. This could lead to the conclusion that ethylene response is important for the suppression of these genes by caterpillars.
MtEBP/ MtEFRs are the transcription factors that regulate the expression of ethylene responsive and stress related genes (Atallah et al., 2008). When these TFs bind to the GCC-box consensus sequence element in the promoter region of ethylene-dependent genes, they lead to activation or repression of these genes (Fujimoto et al., 2000). Apart from ethylene, these ERFs are also responsive to another stress related hormones, like auxin. No herbivory-specific effect was observed in the expression of MtEBP in the wild type as well as skl mutant of M. truncatula. Together with this and no difference in the induction of gene encoding ACC oxidase, an enzyme involved in ethylene biosynthesis, by herbivores could infer that caterpillars do not elicit ethylene pathway in M. truncatula.