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.
Highlights
Plants face constant attacks by a wide array of microorganisms including bacteria, fungi and oomycetes
To establish an inventory of the RxLR effector secretome of Hyaloperonospora arabidopsidis (Hpa), we scanned the draft genome of Emoy2 for all possible open reading frames (ORFs) encoding putative proteins longer than 100 amino acids. We searched these sequences for the presence of signal peptides and from those we extracted gene models carrying RxLR-like motifs (RxLR/Q; RxL) with the sequence and positional constrains defined in Figure 1
When we plotted the percentage of callose suppression of each of the effectors in these groups, we found 77.7% of HaRxLs candidate effectors with a positive effect on Pst-LUX growth in Col-0 were able to suppress callose deposition, while only 3.2% of those decreasing Pst-LUX growth could reduce callose levels (Figure 4 D)
Summary
Plants face constant attacks by a wide array of microorganisms including bacteria, fungi and oomycetes. Obligate biotrophic pathogens are interesting because they can effectively evade or suppress host recognition, thwarting host defenses and enabling pathogen growth and reproduction [1]. Plant disease is rare because plants activate a multilayered defense to most potential pathogens [2]. Conserved molecules, called pathogen (or microbe)associated molecular patterns (PAMPs), are recognized by the plants via pattern recognition receptor proteins (PRRs) [3,4]. This interaction results in pattern-triggered immunity (PTI). Successful pathogens target effector proteins to the host cell cytoplasm to Author Summary
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