RESUMEN
Plant nucleotide-binding leucine-rich repeat receptors (NLRs) act as intracellular sensors for pathogen-derived effector proteins and trigger an immune response, frequently resulting in the hypersensitive cell death response (HR) of the infected host cell. The wheat (Triticum aestivum) NLR Pm2 confers resistance against the fungal pathogen Blumeria graminis f. sp. tritici (Bgt) if the isolate contains the specific RNase-like effector AvrPm2. We identified and isolated seven new Pm2 alleles (Pm2e-i) in the wheat D-genome ancestor Aegilops tauschii and two new natural AvrPm2 haplotypes from Bgt. Upon transient co-expression in Nicotiana benthamiana, we observed a variant-specific HR of the Pm2 variants Pm2a and Pm2i towards AvrPm2 or its homolog from the AvrPm2 effector family, BgtE-5843, respectively. Through the introduction of naturally occurring non-synonymous single nucleotide polymorphisms and structure-guided mutations, we identified single amino acids in both the wheat NLR Pm2 and the fungal effector proteins AvrPm2 and BgtE-5843 responsible for the variant-specific HR of the Pm2 variants. Exchanging these amino acids led to a modified HR of the Pm2-AvrPm2 interaction and allowed the identification of the effector head epitope, a 20-amino-acid long unit of AvrPm2 involved in the HR. Swapping of the AvrPm2 head epitope to the non-HR-triggering AvrPm2 family member BgtE-5846 led to gain of a HR by Pm2a. Our study presents a molecular approach to identify crucial effector surface structures involved in the HR and demonstrates that natural and induced diversity in an immune receptor and its corresponding effectors can provide the basis for understanding and modifying NLR-effector specificity.
Asunto(s)
Aegilops/genética , Ascomicetos/genética , Proteínas Fúngicas/metabolismo , Proteínas NLR/metabolismo , Enfermedades de las Plantas/inmunología , Proteínas de Plantas/metabolismo , Triticum/genética , Alelos , Aminoácidos/metabolismo , Ascomicetos/fisiología , Resistencia a la Enfermedad , Proteínas Fúngicas/genética , Variación Genética , Interacciones Huésped-Patógeno , Mutación , Proteínas NLR/genética , Enfermedades de las Plantas/microbiología , Inmunidad de la Planta , Proteínas de Plantas/genética , Nicotiana/genética , Nicotiana/fisiología , Triticum/inmunología , Triticum/microbiologíaRESUMEN
Plant genomes have evolved several evolutionary mechanisms to tolerate and make use of transposable elements (TEs). Of these, transposon domestication into cis-regulatory and microRNA (miRNA) sequences is proposed to contribute to abiotic/biotic stress adaptation in plants. The wheat genome is derived at 85% from TEs, and contains thousands of miniature inverted-repeat transposable elements (MITEs), whose sequences are particularly prone for domestication into miRNA precursors. In this study, we investigate the contribution of TEs to the wheat small RNA immune response to the lineage-specific, obligate powdery mildew pathogen. We show that MITEs of the Mariner superfamily contribute the largest diversity of miRNAs to the wheat immune response. In particular, MITE precursors of miRNAs are wide-spread over the wheat genome, and highly conserved copies are found in the Lr34 and QPm.tut-4A mildew resistance loci. Our work suggests that transposon domestication is an important evolutionary force driving miRNA functional innovation in wheat immunity.
Asunto(s)
Elementos Transponibles de ADN , MicroARNs/genética , Sitios de Carácter Cuantitativo , Triticum/crecimiento & desarrollo , Adaptación Biológica , Resistencia a la Enfermedad , Domesticación , Evolución Molecular , Dosificación de Gen , Variación Genética , ARN de Planta/genética , Triticum/genética , Triticum/microbiologíaRESUMEN
The wheat Pm3 resistance gene against the powdery mildew pathogen occurs as an allelic series encoding functionally different immune receptors which induce resistance upon recognition of isolate-specific avirulence (AVR) effectors from the pathogen. Here, we describe the identification of five effector proteins from the mildew pathogens of wheat, rye, and the wild grass Dactylis glomerata, specifically recognized by the PM3B, PM3C and PM3D receptors. Together with the earlier identified AVRPM3A2/F2, the recognized AVRs of PM3B/C, (AVRPM3B2/C2), and PM3D (AVRPM3D3) belong to a large group of proteins with low sequence homology but predicted structural similarities. AvrPm3b2/c2 and AvrPm3d3 are conserved in all tested isolates of wheat and rye mildew, and non-host infection assays demonstrate that Pm3b, Pm3c, and Pm3d are also restricting the growth of rye mildew on wheat. Furthermore, divergent AVR homologues from non-adapted rye and Dactylis mildews are recognized by PM3B, PM3C, or PM3D, demonstrating their involvement in host specificity.
Asunto(s)
Ascomicetos/fisiología , Proteínas Fúngicas/inmunología , Especificidad del Huésped , Enfermedades de las Plantas/inmunología , Proteínas de Plantas/inmunología , Triticum/inmunología , Ascomicetos/aislamiento & purificación , Ascomicetos/patogenicidad , Dactylis/microbiología , Resistencia a la Enfermedad/inmunología , Grano Comestible/inmunología , Grano Comestible/microbiología , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Genoma Fúngico , Estudio de Asociación del Genoma Completo , Proteínas NLR/inmunología , Proteínas NLR/metabolismo , Enfermedades de las Plantas/microbiología , Proteínas de Plantas/metabolismo , Plantas Modificadas Genéticamente , Secale/microbiología , Nicotiana/genética , Nicotiana/microbiología , Triticum/microbiologíaRESUMEN
Recognition of the AVRPM3A2/F2 avirulence protein from powdery mildew by the wheat PM3A/F immune receptor induces a hypersensitive response after co-expression in Nicotiana benthamiana. The molecular determinants of this interaction and how they shape natural AvrPm3a2/f2 allelic diversity are unknown. We sequenced the AvrPm3a2/f2 gene in a worldwide collection of 272 mildew isolates. Using the natural polymorphisms of AvrPm3a2/f2 as well as sequence information from related gene family members, we tested 85 single-residue-altered AVRPM3A2/F2 variants with PM3A, PM3F and PM3FL456P/Y458H (modified for improved signaling) in Nicotiana benthamiana for effects on recognition. An intact AvrPm3a2/f2 gene was found in all analyzed isolates and the protein variant recognized by PM3A/F occurred globally at high frequencies. Single-residue alterations in AVRPM3A2/F2 mostly disrupted, but occasionally enhanced, the recognition response by PM3A, PM3F and PM3FL456P/Y458H . Residues enhancing hypersensitive responses constituted a protein domain separate from both naturally occurring polymorphisms and positively selected residues of the gene family. These results demonstrate the utility of using gene family sequence diversity to screen residues for their role in recognition. This approach identified a putative interaction surface in AVRPM3A2/F2 not polymorphic in natural alleles. We conclude that molecular mechanisms besides recognition drive AvrPm3a2/f2 diversification.
Asunto(s)
Ascomicetos/patogenicidad , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Enfermedades de las Plantas/microbiología , Receptores Inmunológicos/metabolismo , Triticum/microbiología , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Ascomicetos/genética , Ascomicetos/aislamiento & purificación , Secuencia Conservada , Proteínas Fúngicas/genética , Regulación Fúngica de la Expresión Génica , Geografía , Mutación/genética , Fenotipo , Proteínas de Plantas/metabolismo , Polimorfismo Genético , Dominios Proteicos , Relación Estructura-Actividad , VirulenciaRESUMEN
In cereals, several mildew resistance genes occur as large allelic series; for example, in wheat (Triticum aestivum and Triticum turgidum), 17 functional Pm3 alleles confer agronomically important race-specific resistance to powdery mildew (Blumeria graminis). The molecular basis of race specificity has been characterized in wheat, but little is known about the corresponding avirulence genes in powdery mildew. Here, we dissected the genetics of avirulence for six Pm3 alleles and found that three major Avr loci affect avirulence, with a common locus_1 involved in all AvrPm3-Pm3 interactions. We cloned the effector gene AvrPm3(a2/f2) from locus_2, which is recognized by the Pm3a and Pm3f alleles. Induction of a Pm3 allele-dependent hypersensitive response in transient assays in Nicotiana benthamiana and in wheat demonstrated specificity. Gene expression analysis of Bcg1 (encoded by locus_1) and AvrPm3 (a2/f2) revealed significant differences between isolates, indicating that in addition to protein polymorphisms, expression levels play a role in avirulence. We propose a model for race specificity involving three components: an allele-specific avirulence effector, a resistance gene allele, and a pathogen-encoded suppressor of avirulence. Thus, whereas a genetically simple allelic series controls specificity in the plant host, recognition on the pathogen side is more complex, allowing flexible evolutionary responses and adaptation to resistance genes.