Multiple Candidate Effectors from the Oomycete Pathogen Hyaloperonospora arabidopsidis Suppress Host Plant Immunity

Abstract

Oomycete pathogens cause diverse plant diseases. To successfully colonize their hosts, they deliver a suite of effector proteins that can attenuate plant defenses. In the oomycete downy mildews, effectors carry a signal peptide and an RxLR motif. Hyaloperonospora arabidopsidis (Hpa) causes downy mildew on the model plant Arabidopsis thaliana (Arabidopsis). We investigated if candidate effectors predicted in the genome sequence of Hpa isolate Emoy2 (HaRxLs) were able to manipulate host defenses in different Arabidopsis accessions. We developed a rapid and sensitive screening method to test HaRxLs by delivering them via the bacterial type-three secretion system (TTSS) of Pseudomonas syringae pv tomato DC3000-LUX (Pst-LUX) and assessing changes in Pst-LUX growth in planta on 12 Arabidopsis accessions. The majority (∼70%) of the 64 candidates tested positively contributed to Pst-LUX growth on more than one accession indicating that Hpa virulence likely involves multiple effectors with weak accession-specific effects. Further screening with a Pst mutant (ΔCEL) showed that HaRxLs that allow enhanced Pst-LUX growth usually suppress callose deposition, a hallmark of pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI). We found that HaRxLs are rarely strong avirulence determinants. Although some decreased Pst-LUX growth in particular accessions, none activated macroscopic cell death. Fewer HaRxLs conferred enhanced Pst growth on turnip, a non-host for Hpa, while several reduced it, consistent with the idea that turnip's non-host resistance against Hpa could involve a combination of recognized HaRxLs and ineffective HaRxLs. We verified our results by constitutively expressing in Arabidopsis a sub-set of HaRxLs. Several transgenic lines showed increased susceptibility to Hpa and attenuation of Arabidopsis PTI responses, confirming the HaRxLs' role in Hpa virulence. This study shows TTSS screening system provides a useful tool to test whether candidate effectors from eukaryotic pathogens can suppress/trigger plant defense mechanisms and to rank their effectiveness prior to subsequent mechanistic investigation.

Specific adaptation of Ustilaginoidea virens in occupying host florets revealed by comparative and functional genomics

“…Putative effector genes (without signal peptide regions) of U. virens were amplified with the primer sets listed in Supplementary Table 31. The PCR products were digested with the corresponding restriction emzymes and subcloned into pEDV 70 . Burkholderia glumae competent cells were prepared as described with minor modifications 43 .…”

Mechanisms of Nuclear Suppression of Host Immunity by Effectors from the Arabidopsis Downy Mildew Pathogen Hyaloperonospora arabidopsidis (Hpa)

“…When expressed in roots, SP7 leads to higher mycorrhization, whereas expression of SP7 in the necrotrophic fungus Magnaporthe oryzae attenuates root decay symptoms (Kloppholz et al 2011). Recent development of the heterologous system to study effectors from biotrophic pathogens enabled the discovery and the functional analysis of a biotroph effector repertoire (Fabro et al 2011), revealing effector candidates that enhance plant susceptibility to pathogens that are mainly localized to the plant cell nucleus (Caillaud et al 2012). Functional analysis of such nuclear HaRxLs effectors will allow dissection of the components of the nuclear plant immune system targeted by biotrophic pathogens to promote long-term coexistence with their hosts.…”

“…For this reason, we analyzed stable effector expression in planta, particularly for studying Arabidopsis-compatible pathosystems. Stable effector expression in planta allows us to test whether the ectopic expression of a given effector affects plant susceptibility toward the corresponding pathogen (Bozkurt et al 2011;Fabro et al 2011;Caillaud et al 2012) or even other pathogens (van Esse et al 2008;Hewezi et al 2010;Bouwmeester et al 2011;Fabro et al 2011). Using this approach, effectors from Hpa, including ATR13, were shown to suppress plant defenses, notably PAMP-induced ROS burst and callose deposition, which are typical indicators of plant immune response activation (Sohn et al 2007;Fabro et al 2011).…”

“…HaRxLL3b induced defects in leaf curvature whereas HaRxL73 expression leads to albino and early flowering plants. HaRxL106 and HaRxLL60 modify the shape of the leaves that become either elongated and darker or serrated and smaller, respectively (Fabro et al 2011). HaRxL36 causes flowers with variable numbers of petals, whereas HaRxL2 provokes twisted plant organs (Fig.…”

“…In parallel, a collection of "HaRxL-EDV" from Hpa Emoy2 was screened for bacterial virulence on several Arabidopsis accessions (Table 1). We observed that variable HaRxLs, such as ATR1 and ATR13, performed differently on selected Arabidopsis accessions, whereas conserved "core" HaRxLs, such as HaRxL62, enhance plant susceptibility to Pst in all of the Arabidopsis accessions (Fabro et al 2011). Taking advantage of the HaRxL collection created by Fabro et al (2011), we designed a fluorescence-imaging-based screen in planta to assess their subcellular localization.…”