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.