Véronique Chalvon

The Rice Resistance Protein Pair RGA4/RGA5 Recognizes the Magnaporthe oryzae Effectors AVR-Pia and AVR1-CO39 by Direct Binding

This work shows that the rice NB-LRR protein pair, RGA4 and RGA5-A, has a dual recognition specificity and detects the Magnaporthe oryzae effectors AVR1-CO39 and AVR-Pia, which have unrelated sequences. Recognition seems to be mediated by direct binding of the Avr proteins to a novel non-LRR domain of RGA5-A also present in the Avr binding domain of the rice resistance protein Pik-1. Resistance (R) proteins recognize pathogen avirulence (Avr) proteins by direct or indirect binding and are multidomain proteins generally carrying a nucleotide binding (NB) and a leucine-rich repeat (LRR) domain. Two NB-LRR protein-coding genes from rice (Oryza sativa), RGA4 and RGA5, were found to be required for the recognition of the Magnaporthe oryzae effector AVR1-CO39. RGA4 and RGA5 also mediate recognition of the unrelated M. oryzae effector AVR-Pia, indicating that the corresponding R proteins possess dual recognition specificity. For RGA5, two alternative transcripts, RGA5-A and RGA5-B, were identified. Genetic analysis showed that only RGA5-A confers resistance, while RGA5-B is inactive. Yeast two-hybrid, coimmunoprecipitation, and fluorescence resonance energy transfer–fluorescence lifetime imaging experiments revealed direct binding of AVR-Pia and AVR1-CO39 to RGA5-A, providing evidence for the recognition of multiple Avr proteins by direct binding to a single R protein. Direct binding seems to be required for resistance as an inactive AVR-Pia allele did not bind RGA5-A. A small Avr interaction domain with homology to the Avr recognition domain in the rice R protein Pik-1 was identified in the C terminus of RGA5-A. This reveals a mode of Avr protein recognition through direct binding to a novel, non-LRR interaction domain.

The NB‐LRR proteins RGA4 and RGA5 interact functionally and physically to confer disease resistance

Plant resistance proteins of the class of nucleotide‐binding and leucine‐rich repeat domain proteins (NB‐LRRs) are immune sensors which recognize pathogen‐derived molecules termed avirulence (AVR) proteins. We show that RGA4 and RGA5, two NB‐LRRs from rice, interact functionally and physically to mediate resistance to the fungal pathogen Magnaporthe oryzae and accomplish different functions in AVR recognition. RGA4 triggers an AVR‐independent cell death that is repressed in the presence of RGA5 in both rice protoplasts and Nicotiana benthamiana. Upon recognition of the pathogen effector AVR‐Pia by direct binding to RGA5, repression is relieved and cell death occurs. RGA4 and RGA5 form homo‐ and hetero‐complexes and interact through their coiled‐coil domains. Localization studies in rice protoplast suggest that RGA4 and RGA5 localize to the cytosol. Upon recognition of AVR‐Pia, neither RGA4 nor RGA5 is re‐localized to the nucleus. These results establish a model for the interaction of hetero‐pairs of NB‐LRRs in plants: RGA4 mediates cell death activation, while RGA5 acts as a repressor of RGA4 and as an AVR receptor.

Preformed expression of defense is a hallmark of partial resistance to rice blast fungal pathogen Magnaporthe oryzae

BackgroundPartial resistance to plant pathogens is extensively used in breeding programs since it could contribute to resistance durability. Partial resistance often builds up during plant development and confers quantitative and usually broad-spectrum resistance. However, very little is known on the mechanisms underlying partial resistance. Partial resistance is often explained by poorly effective induction of plant defense systems. By exploring rice natural diversity, we asked whether expression of defense systems before infection could explain partial resistance towards the major fungal pathogen Magnaporthe oryzae. The constitutive expression of 21 defense-related genes belonging to the defense system was monitored in 23 randomly sampled rice cultivars for which partial resistance was measured.ResultsWe identified a strong correlation between the expression of defense-related genes before infection and partial resistance. Only a weak correlation was found between the induction of defense genes and partial resistance. Increasing constitutive expression of defense-related genes also correlated with the establishment of partial resistance during plant development. Some rice genetic sub-groups displayed a particular pattern of constitutive expression, suggesting a strong natural polymorphism for constitutive expression of defense. Constitutive levels of hormones like salicylic acid and ethylene cannot explain constitutive expression of defense. We could identify an area of the genome that contributes to explain both preformed defense and partial resistance.ConclusionThese results indicate that constitutive expression of defense-related genes is likely responsible for a large part of partial resistance in rice. The finding of this preformed defense system should help guide future breeding programs and open the possibility to identify the molecular mechanisms behind partial resistance.

Cytokinin Production by the Rice Blast Fungus Is a Pivotal Requirement for Full Virulence

Plants produce cytokinin (CK) hormones for controlling key developmental processes like source/sink distribution, cell division or programmed cell-death. Some plant pathogens have been shown to produce CKs but the function of this mimicry production by non-tumor inducing pathogens, has yet to be established. Here we identify a gene required for CK biosynthesis, CKS1, in the rice blast fungus Magnaporthe oryzae. The fungal-secreted CKs are likely perceived by the plant during infection since the transcriptional regulation of rice CK-responsive genes is altered in plants infected by the mutants in which CKS1 gene was deleted. Although cks1 mutants showed normal in vitro growth and development, they were severely affected for in planta growth and virulence. Moreover, we showed that the cks1 mutant triggered enhanced induction of plant defenses as manifested by an elevated oxidative burst and expression of defense-related markers. In addition, the contents of sugars and key amino acids for fungal growth were altered in and around the infection site by the cks1 mutant in a different manner than by the control strain. These results suggest that fungal-derived CKs are key effectors required for dampening host defenses and affecting sugar and amino acid distribution in and around the infection site.

Recognition of the Magnaporthe oryzae Effector AVR-Pia by the Decoy Domain of the Rice NLR Immune Receptor RGA5

The Magnaporthe oryzae effector AVR-Pia interacts with the decoy domain of the rice NLR immune receptor RGA5 through a defined surface area, and this interaction is required for effector recognition. Nucleotide binding domain and leucine-rich repeat proteins (NLRs) are important receptors in plant immunity that allow recognition of pathogen effectors. The rice (Oryza sativa) NLR RGA5 recognizes the Magnaporthe oryzae effector AVR-Pia through direct interaction. Here, we gained detailed insights into the molecular and structural bases of AVR-Pia-RGA5 interaction and the role of the RATX1 decoy domain of RGA5. NMR titration combined with in vitro and in vivo protein-protein interaction analyses identified the AVR-Pia interaction surface that binds to the RATX1 domain. Structure-informed AVR-Pia mutants showed that, although AVR-Pia associates with additional sites in RGA5, binding to the RATX1 domain is necessary for pathogen recognition but can be of moderate affinity. Therefore, RGA5-mediated resistance is highly resilient to mutations in the effector. We propose a model that explains such robust effector recognition as a consequence, and an advantage, of the combination of integrated decoy domains with additional independent effector-NLR interactions.

Identification of positive and negative regulators of disease resistance to rice blast fungus using constitutive gene expression patterns.

Elevated constitutive expression of components of the defence arsenal is associated with quantitative resistance to the rice blast fungus, a phenomenon called preformed defence. While the role of many disease regulators in inducible defence systems has been extensively studied, little attention has been paid so far to genes that regulate preformed defence. In this study, we show by microarray analysis across rice diversity that the preformed defence phenomenon impacts on a large number of defence-related genes without apparently affecting other biological processes. Using a guilt-by-association strategy, we identified two positive regulators that promote constitutive expression of known defence markers and partial resistance to rice blast. The HSF23 gene encodes for a putative member of the heat shock transcription factor family, while CaMBP encodes for a putative Calmodulin-binding protein. Both HSF23 and CaMBP strongly affect preformed defence and also plant growth. Additionally, we identified the OB-fold gene as a negative regulator of blast resistance, which could be involved in RNA stabilization. The OB-fold mutants do not suffer from obvious developmental defects. Taken together, our results prove that our strategy of combining analysis of gene expression diversity with guilt-by-association is a powerful way to identify disease resistance regulators in rice.

Ectopic activation of the rice NLR heteropair RGA4/RGA5 confers resistance to bacterial blight and bacterial leaf streak diseases.

Bacterial blight (BB) and bacterial leaf streak (BLS) are important diseases in Oryza sativa caused by Xanthomonas oryzae pv. oryzae (Xoo) and Xanthomonas oryzae pv. oryzicola (Xoc), respectively. In both bacteria, transcription activator-like (TAL) effectors are major virulence determinants that act by transactivating host genes downstream of effector-binding elements (EBEs) bound in a sequence-specific manner. Resistance to Xoo is mostly related to the action of TAL effectors, either by polymorphisms that prevent the induction of susceptibility (S) genes or by executor (R) genes with EBEs embedded in their promoter, and that induce cell death and resistance. For Xoc, no resistance sources are known in rice. Here, we investigated whether the recognition of effectors by nucleotide binding and leucine-rich repeat domain immune receptors (NLRs), the most widespread resistance mechanism in plants, is also able to stop BB and BLS. In one instance, transgenic rice lines harboring the AVR1-CO39 effector gene from the rice blast fungus Magnaporthe oryzae, under the control of an inducible promoter, were challenged with transgenic Xoo and Xoc strains carrying a TAL effector designed to transactivate the inducible promoter. This induced AVR1-CO39 expression and triggered BB and BLS resistance when the corresponding Pi-CO39 resistance locus was present. In a second example, the transactivation of an auto-active NLR by Xoo-delivered designer TAL effectors resulted in BB resistance, demonstrating that NLR-triggered immune responses efficiently control Xoo. This forms the foundation for future BB and BLS disease control strategies, whereupon endogenous TAL effectors will target synthetic promoter regions of Avr or NLR executor genes.