RNAi Silencing of Genes for Elicitation or Biosynthesis of 5-Deoxyisoflavonoids Suppresses Race-Specific Resistance and Hypersensitive Cell Death in Phytophthora sojae Infected Tissues

Abstract

Isoflavonoids are thought to play an important role in soybean (Glycine max) resistance to Phytophthora sojae. This was addressed by silencing two genes for their biosynthesis and a third gene controlling their elicitation. Silencing of genes for isoflavone synthase (IFS) or chalcone reductase (CHR) was achieved in soybean roots through an Agrobacterium rhizogenes-mediated RNAi approach. Effectiveness of silencing was followed both by quantitative reverse transcriptase-polymerase chain reaction and high-performance liquid chromatography analyses. Silencing either IFS or CHR led to a breakdown of Rps-mediated resistance to race 1 of P. sojae in 'W79' (Rps 1c) or 'W82' (Rps 1k) soybean. Loss of resistance was accompanied by suppression of hypersensitive (HR) cell death in both cultivars and suppression of cell death-associated activation of hydrogen peroxide and peroxidase. The various results suggest that the 5-deoxyisoflavonoids play a critical role in the establishment of cell death and race-specific resistance. The P. sojae cell wall glucan elicitor, a potent elicitor of 5-deoxyisoflavonoids, triggered a cell death response in roots that was also suppressed by silencing either CHR or IFS. Furthermore, silencing of the elicitor-releasing endoglucanase (PR-2) led to a loss of HR cell death and race-specific resistance to P. sojae and also to a loss of isoflavone and cell death responses to cell wall glucan elicitor. Taken together, these results suggest that in situ release of active fragments from a general resistance elicitor (pathogen-associated molecular pattern) is necessary for HR cell death in soybean roots carrying resistance genes at the Rps 1 locus, and that this cell death response is mediated through accumulations of the 5-deoxyisoflavones.

Nature’s assembly line: biosynthesis of simple phenylpropanoids and polyketides

“…Disruption of IFS gene expression in soybean hairy root composite plants using RNA-mediated interference significantly reduces daidzein and genistein levels . Similarly, examination of TILLING populations with mutated IFS genes in Medicago truncatula indicates that some legumes can survive without isoflavones; however, the isoflavone-null soybean roots showed increased susceptibility to fungal infections (Graham et al, 2007). This result highlights the important role that isoflavonoids play in maintaining plant health.…”

The Nematode Resistance Allele at the rhg1 Locus Alters the Proteome and Primary Metabolism of Soybean Roots

“…The enzyme acts directly in plant disease resistance through digesting the cell wall of the invading pathogen (Bishop et al, 2005). The released cell wall fragments may also act as elicitors or in resistance, resulting in an accentuated response (Graham et al, 2007).…”

“…Endoglucanases have been reported to be activated by both biotic and abiotic stresses. The pathogenesis protein PR2 is a b-1,3-endoglucanase (Graham et al, 2003(Graham et al, , 2007. The enzyme acts directly in plant disease resistance through digesting the cell wall of the invading pathogen (Bishop et al, 2005).…”

Application of somatic embryos to rapid and reliable analysis of soybean seed components by RNA interference-mediated gene silencing

“…RNAi-mediated gene silencing is therefore a potentially effective tool for characterization of gene function in soybean because of its ability to overcome functional redundancy of duplicated genes (Lawrence and Pikaard 2003;Miki et al 2005;Travella et al 2006). The high efficiency of Agrobacterium rhizogenes-mediated transformation has resulted in the widespread application of this approach to highthroughput analysis of cellular and biochemical processes in roots by RNAi-mediated silencing (Graham et al 2007;Lee et al 2005;Li et al 2010;Subramanian et al 2005). Although hairy roots are not suitable for the study of seed-specific traits because whole plants or embryos cannot be recovered from them, suppression of the isoflavone synthase gene by this approach was apparent not only in the emerged transgenic hairy roots but also in cotyledons at a position distal to the site of RNAi transformation by A. rhizogenes (Subramanian et al 2005).…”