Host-Induced Gene Silencing in Barley Powdery Mildew Reveals a Class of Ribonuclease-Like Effectors

Abstract

Obligate biotrophic pathogens of plants must circumvent or counteract defenses to guarantee accommodation inside the host. To do so, they secrete a variety of effectors that regulate host immunity and facilitate the establishment of pathogen feeding structures called haustoria. The barley powdery mildew fungus Blumeria graminis f. sp. hordei produces a large number of proteins predicted to be secreted from haustoria. Fifty of these Blumeria effector candidates (BEC) were screened by host-induced gene silencing (HIGS), and eight were identified that contribute to infection. One shows similarity to β-1,3 glucosyltransferases, one to metallo-proteases, and two to microbial secreted ribonucleases; the remainder have no similarity to proteins of known function. Transcript abundance of all eight BEC increases dramatically in the early stages of infection and establishment of haustoria, consistent with a role in that process. Complementation analysis using silencing-insensitive synthetic cDNAs demonstrated that the ribonuclease-like BEC 1011 and 1054 are bona fide effectors that function within the plant cell. BEC1011 specifically interferes with pathogen-induced host cell death. Both are part of a gene superfamily unique to the powdery mildew fungi. Structural modeling was consistent, with BEC1054 adopting a ribonuclease-like fold, a scaffold not previously associated with effector function.

Mosaic genome structure of the barley powdery mildew pathogen and conservation of transcriptional programs in divergent hosts

“…Therefore, an active secretion mechanism has been proposed that exports these CSEPs from the haustorium (22). Furthermore, although detailed biological roles of most CSEPs have not been documented, host-induced gene silencing (23) of some CSEPs suggested their role in host-defense suppression (24,25). Notably, the CSEP repertoire of Bgh is strikingly different from those of the distantly related Golovinomyces orontii and Erysiphe pisi powdery mildews, suggesting specific adaptations to their respective host plants (15).…”

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“…Host induced gene silencing (HIGS) is a relatively new approach for controlling plant pathogens that relies on RNA interference to target the expression of essential pathogen genes. This strategy has been used to target a wide range of pathogen types including insects (reviews by Baum et al 2007;Huvenne and Smagghe 2010), nematodes (reviewed by Huang et al 2006;Yadav et al 2006;Fairbairn et al 2007;Sindhu et al 2009), fungi (Nowara et al 2010;Tinoco et al 2010;Yin et al 2011;Zhang et al 2012;Koch et al 2013;Panwar et al 2013;Pliego et al 2013;Ghag et al 2014), parasitic weeds (Tomilov et al 2008), and oomycetes (Govindarajulu et al 2014;VegaArreguin et al 2014;Jahan et al 2015). Pathogen effectors essential for virulence or Bhousekeeping^genes necessary for normal pathogen growth are typically the targets for HIGS.…”

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“…HIGS phenomenon was first observed in barley-Bgh interaction (Nowara et al 2010) and now the idea has provided a method to investigate the role of pathogen genes (Pliego et al 2013). It also suggests a potential approach to engineering plants for resistance to PMs (Nowara et al 2010).…”

“…In incompatible interactions (e.g., presence of Mla1 gene) the CSEPs expression decreases following a transcriptional reprogramming in barley epidermal cells and at the onset of cell death in those cells, suggesting a defence mechanism by which host suppresses production or secretion of pathogen's effectors (Hacquard, et al 2013). Functional analyses of 50 of these CSEPs showed that silencing of eight of them, which are similar to glucosyltransferases, metalloproteases, and microbial secreted ribonucleases, inside the pathogen compromised disease development (Pliego et al 2013). The ribonuclease-like effectors probably interfere with programed cell death in the host cells and, therefore, help establishment of pathogenicity (Pliego et al 2013).…”

“…Functional analyses of 50 of these CSEPs showed that silencing of eight of them, which are similar to glucosyltransferases, metalloproteases, and microbial secreted ribonucleases, inside the pathogen compromised disease development (Pliego et al 2013). The ribonuclease-like effectors probably interfere with programed cell death in the host cells and, therefore, help establishment of pathogenicity (Pliego et al 2013). In another study, it was shown that one of the CSEPs (CSEP0055) interacts with apoplastic pathogenesis-related proteins of barley, including PR17, and thereby suppresses the host defence (Zhang et al 2012).…”