The Arabidopsis WRR4A and WRR4B paralogous NLR proteins both confer recognition of multiple Albugo candida effectors.

The oomycete Albugo candida causes white blister rust, an important disease of Brassica crops. Distinct races of A. candida are defined by their capacity to infect different host plant species. Each A. candida race encodes secreted proteins with a CX2 CX5 G ('CCG') motif that are polymorphic and show presence/absence variation, and are therefore candidate effectors. The White Rust Resistance 4 (WRR4) locus in Arabidopsis thaliana accession Col-0 contains three genes that encode intracellular nucleotide-binding domain leucine-rich repeat immune receptors. The Col-0 alleles of WRR4A and WRR4B confer resistance to multiple A. candida races, although both WRR4A and WRR4B can be overcome by the Col-0-virulent race 4 isolate AcEx1. Comparison of CCG candidate effectors in avirulent and virulent races, and transient co-expression of CCG effectors from four A. candida races in Nicotiana sp. or A. thaliana, revealed CCG effectors that trigger WRR4A- or WRR4B-dependent hypersensitive responses. We found eight WRR4A-recognised CCGs and four WRR4B-recognised CCGs, the first recognised proteins from A. candida for which the cognate immune receptors in A. thaliana are known. This multiple recognition capacity potentially explains the broad-spectrum resistance to several A. candida races conferred by WRR4 paralogues. We further show that of five tested CCGs, three confer enhanced disease susceptibility when expressed in planta, consistent with A. candida CCG proteins being effectors.

Leptosphaeria maculans Effector Protein AvrLm1 Modulates Plant Immunity by Enhancing MAP Kinase 9 Phosphorylation.

Leptosphaeria maculans, the causal agent of blackleg disease in canola (Brassica napus), secretes an array of effectors into the host to overcome host defense. Here we present evidence that the L. maculans effector protein AvrLm1 functions as a virulence factor by interacting with the B. napus mitogen-activated protein (MAP) kinase 9 (BnMPK9), resulting in increased accumulation and enhanced phosphorylation of the host protein. Transient expression of BnMPK9 in Nicotiana benthamiana induces cell death, and this phenotype is enhanced in the presence of AvrLm1, suggesting that induction of cell death due to enhanced accumulation and phosphorylation of BnMPK9 by AvrLm1 supports the initiation of necrotrophic phase of L. maculans infection. Stable expression of BnMPK9 in B. napus perturbs hormone signaling, notably salicylic acid response genes, to facilitate L. maculans infection. Our findings provide evidence that a MAP kinase is directly targeted by a fungal effector to modulate plant immunity.

Multi-environment QTL studies suggest a role for cysteine-rich protein kinase genes in quantitative resistance to blackleg disease in Brassica napus.

BACKGROUND: Resistance to the blackleg disease of Brassica napus (canola/oilseed rape), caused by the hemibiotrophic fungal pathogen Leptosphaeria maculans, is determined by both race-specific resistance (R) genes and quantitative resistance loci (QTL), or adult-plant resistance (APR). While the introgression of R genes into breeding material is relatively simple, QTL are often detected sporadically, making them harder to capture in breeding programs. For the effective deployment of APR in crop varieties, resistance QTL need to have a reliable influence on phenotype in multiple environments and be well defined genetically to enable marker-assisted selection (MAS). RESULTS: Doubled-haploid populations produced from the susceptible B. napus variety Topas and APR varieties AG-Castle and AV-Sapphire were analysed for resistance to blackleg in two locations over 3 and 4 years, respectively. Three stable QTL were detected in each population, with two loci appearing to be common to both APR varieties. Physical delineation of three QTL regions was sufficient to identify candidate defense-related genes, including a cluster of cysteine-rich receptor-like kinases contained within a 49 gene QTL interval on chromosome A01. Individual L. maculans isolates were used to define the physical intervals for the race-specific R genes Rlm3 and Rlm4 and to identify QTL common to both field studies and the cotyledon resistance response. CONCLUSION: Through multi-environment QTL analysis we have identified and delineated four significant and stable QTL suitable for MAS of quantitative blackleg resistance in B. napus, and identified candidate genes which potentially play a role in quantitative defense responses to L. maculans.

Genome-wide transcriptomic analyses provide insights into the lifestyle transition and effector repertoire of Leptosphaeria maculans during the colonization of Brassica napus seedlings.

Molecular interaction between the causal agent of blackleg disease, Leptosphaeria maculans (Lm), and its host, Brassica napus, is largely unknown. We applied a deep RNA-sequencing approach to gain insight into the pathogenicity mechanisms of Lm and the defence response of B. napus. RNA from the infected susceptible B. napus cultivar Topas DH16516, sampled at 2-day intervals (0-8 days), was sequenced and used for gene expression profiling. Patterns of gene expression regulation in B. napus showed multifaceted defence responses evident by the differential expression of genes encoding the pattern recognition receptor CERK1 (chitin elicitor receptor kinase 1), receptor like proteins and WRKY transcription factors. The up-regulation of genes related to salicylic acid and jasmonic acid at the initial and late stages of infection, respectively, provided evidence for the biotrophic and necrotrophic life stages of Lm during the infection of B. napus cotyledons. Lm transition from biotrophy to necrotropy was also supported by the expression function of Lm necrosis and ethylene-inducing (Nep-1)-like peptide. Genes encoding polyketide synthases and non-ribosomal peptide synthetases, with potential roles in pathogenicity, were up-regulated at 6-8 days after inoculation. Among other plant defence-related genes differentially regulated in response to Lm infection were genes involved in the reinforcement of the cell wall and the production of glucosinolates. Dual RNA-sequencing allowed us to define the Lm candidate effectors expressed during the infection of B. napus. Several candidate effectors suppressed Bax-induced cell death when transiently expressed in Nicotiana benthamaina leaves.

The receptor-like kinase SOBIR1 interacts with Brassica napus LepR3 and is required for Leptosphaeria maculans AvrLm1-triggered immunity.

The fungus Leptosphaeria maculans (L. maculans) is the causal agent of blackleg disease of canola/oilseed rape (Brassica napus) worldwide. We previously reported cloning of the B. napus blackleg resistance gene, LepR3, which encodes a receptor-like protein. LepR3 triggers localized cell death upon recognition of its cognate Avr protein, AvrLm1. Here, we exploited the Nicotiana benthamiana model plant to investigate the recognition mechanism of AvrLm1 by LepR3. Co-expression of the LepR3/AvrLm1 gene pair in N. benthamiana resulted in development of a hypersensitive response (HR). However, a truncated AvrLm1 lacking its indigenous signal peptide was compromised in its ability to induce LepR3-mediated HR, indicating that AvrLm1 is perceived by LepR3 extracellularly. Structure-function analysis of the AvrLm1 protein revealed that the C-terminal region of AvrLm1 was required for LepR3-mediated HR in N. benthamiana and for resistance to L. maculans in B. napus. LepR3 was shown to be physically interacting with the B. napus receptor like kinase, SOBIR1 (BnSOBIR1). Silencing of NbSOBIR1 or NbSERK3 (BAK1) compromised LepR3-AvrLm1-dependent HR in N. benthamiana, suggesting that LepR3-mediated resistance to L. maculans in B. napus requires SOBIR1 and BAK1/SERK3. Using this model system, we determined that BnSOBIR1 and SERK3/BAK1 are essential partners in the LepR3 signaling complex and were able to define the AvrLm1 effector domain.

Rapid identification of the Leptosphaeria maculans avirulence gene AvrLm2 using an intraspecific comparative genomics approach.

Five avirulence genes from Leptosphaeria maculans, the causal agent of blackleg of canola (Brassica napus), have been identified previously through map-based cloning. In this study, a comparative genomic approach was used to clone the previously mapped AvrLm2. Given the lack of a presence-absence gene polymorphism coincident with the AvrLm2 phenotype, 36 L. maculans isolates were resequenced and analysed for single-nucleotide polymorphisms (SNPs) in predicted small secreted protein-encoding genes present within the map interval. Three SNPs coincident with the AvrLm2 phenotype were identified within LmCys1, previously identified as a putative effector-coding gene. Complementation of a virulent isolate with LmCys1, as the candidate AvrLm2 allele, restored the avirulent phenotype on Rlm2-containing B. napus lines. AvrLm2 encodes a small cysteine-rich protein with low similarity to other proteins in the public databases. Unlike other avirulence genes, AvrLm2 resides in a small GC island within an AT-rich isochore of the genome, and was never found to be deleted completely in virulent isolates.

TMV-Gate vectors: gateway compatible tobacco mosaic virus based expression vectors for functional analysis of proteins.

Plant viral expression vectors are advantageous for high-throughput functional characterization studies of genes due to their capability for rapid, high-level transient expression of proteins. We have constructed a series of tobacco mosaic virus (TMV) based vectors that are compatible with Gateway technology to enable rapid assembly of expression constructs and exploitation of ORFeome collections. In addition to the potential of producing recombinant protein at grams per kilogram FW of leaf tissue, these vectors facilitate either N- or C-terminal fusions to a broad series of epitope tag(s) and fluorescent proteins. We demonstrate the utility of these vectors in affinity purification, immunodetection and subcellular localisation studies. We also apply the vectors to characterize protein-protein interactions and demonstrate their utility in screening plant pathogen effectors. Given its broad utility in defining protein properties, this vector series will serve as a useful resource to expedite gene characterization efforts.