The downy mildew (Hyaloperonospora parasitica) effector proteins ATR1 and ATR13 trigger RPP1-Nd/WsB-and RPP13-Nddependent resistance, respectively, in Arabidopsis thaliana. To better understand the functions of these effectors during compatible and incompatible interactions of H. parasitica isolates on Arabidopsis accessions, we developed a novel delivery system using Pseudomonas syringae type III secretion via fusions of ATRs to the N terminus of the P. syringae effector protein, AvrRPS4. ATR1 and ATR13 both triggered the hypersensitive response (HR) and resistance to bacterial pathogens in Arabidopsis carrying RPP1-Nd/WsB or RPP13-Nd, respectively, when delivered from P. syringae pv tomato (Pst) DC3000. In addition, multiple alleles of ATR1 and ATR13 confer enhanced virulence to Pst DC3000 on susceptible Arabidopsis accessions. We conclude that ATR1 and ATR13 positively contribute to pathogen virulence inside host cells. Two ATR13 alleles suppressed bacterial PAMP (for Pathogen-Associated Molecular Patterns)-triggered callose deposition in susceptible Arabidopsis when delivered by DC3000 DCEL mutants. Furthermore, expression of another allele of ATR13 in plant cells suppressed PAMP-triggered reactive oxygen species production in addition to callose deposition. Intriguingly, although Wassilewskija (Ws-0) is highly susceptible to H. parasitica isolate Emco5, ATR13 Emco5 when delivered by Pst DC3000 triggered localized immunity, including HR, on Ws-0. We suggest that an additional H. parasitica Emco5 effector might suppress ATR13-triggered immunity.

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.

SummaryA screen was established for mutants in which the plant defence response is de-repressed. The pathogeninducible isochorismate synthase (ICS1) promoter was fused to firefly luciferase (luc) and a homozygous transgenic line generated in which the ICS1:luc fusion is co-regulated with ICS1. This line was mutagenized and M 2 seedlings screened for constitutive ICS1:luc expression (cie). The cie mutants fall into distinct phenotypic classes based on tissue-specific localization of luciferase activity. One mutant, cie1, that shows constitutive luciferase activity specifically in petioles, was chosen for further analysis. In addition to ICS1, PR and other defence-related genes are constitutively expressed in cie1 plants. The cie1 mutant is also characterized by an increased production of conjugated salicylic acid and reactive oxygen intermediates, as well as spontaneous lesion formation, all confined to petiole tissue. Significantly, defences activated in cie1 are sufficient to prevent infection by a virulent isolate of Hyaloperonospora parasitica, and this enhanced resistance response protects petiole tissue alone. Furthermore, cie1-mediated resistance, along with PR gene expression, is abolished in a sid2-1 mutant background, consistent with a requirement for salicylic acid. A positional cloning approach was used to identify cie1, which carries two point mutations in a gene required for cell wall biosynthesis and actin organization, MUR3. A mur3 knockout mutant also resists infection by H. parasitica in its petioles and this phenotype is complemented by transformation with wild-type MUR3. We propose that perturbed cell wall biosynthesis may activate plant defence and provide a rationale for the cie1 and the mur3 knockout phenotypes.

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