Instability of the Magnaporthe oryzae avirulence gene AVR-Pita alters virulence

The avirulence gene AVR-Pita of Magnaporthe oryzae determines the efficacy of the resistance gene Pi-ta in rice. The structures of the AVR-Pita alleles in 39 US isolates of M. oryzae were analyzed using polymerase chain reaction. A series of allele-specific primers were developed from the AVR-Pita gene to examine the presence of AVR-Pita. Orthologous alleles of the AVR-Pita gene were amplified from avirulent isolates. Sequence analysis of five alleles revealed three introns at identical positions in the AVR-Pita gene. All five alleles were predicted to encode metalloprotease proteins highly similar to the AVR-Pita protein. In contrast, the same regions of the AVR-Pita alleles were not amplified in the most virulent isolates, and significant variations of DNA sequence at the AVR-Pita allele were verified by Southern blot analysis. A Pot3 transposon was identified in the DNA region encoding the putative protease motif of the AVR-Pita protein from a field isolate B2 collected from a Pi-ta-containing cultivar Banks. These findings show that transposons can contribute to instability of AVR-Pita and is one molecular mechanism for defeating resistance genes in rice cultivar Banks.     

Multiple translocation of the AVR-Pita effector gene among chromosomes of the rice blast fungus Magnaporthe oryzae and related species

Magnaporthe oryzae is the causal agent of rice blast disease, a devastating problem worldwide. This fungus has caused breakdown of resistance conferred by newly developed commercial cultivars. To address how the rice blast fungus adapts itself to new resistance genes so quickly, we examined chromosomal locations of AVR-Pita, a subtelomeric gene family corresponding to the Pita resistance gene, in various isolates of M. oryzae (including wheat and millet pathogens) and its related species. We found that AVR-Pita (AVR-Pita1 and AVR-Pita2) is highly variable in its genome location, occurring in chromosomes 1, 3, 4, 5, 6, 7, and supernumerary chromosomes, particularly in rice-infecting isolates. When expressed in M. oryzae, most of the AVR-Pita homologs could elicit Pita-mediated resistance, even those from non-rice isolates. AVR-Pita was flanked by a retrotransposon, which presumably contributed to its multiple translocation across the genome. On the other hand, family member AVR-Pita3, which lacks avirulence activity, was stably located on chromosome 7 in a vast majority of isolates. These results suggest that the diversification in genome location of AVR-Pita in the rice isolates is a consequence of recognition by Pita in rice. We propose a model that the multiple translocation of AVR-Pita may be associated with its frequent loss and recovery mediated by its transfer among individuals in asexual populations. This model implies that the high mobility of AVR-Pita is a key mechanism accounting for the rapid adaptation toward Pita. Dynamic adaptation of some fungal plant pathogens may be achieved by deletion and recovery of avirulence genes using a population as a unit of adaptation.     

稻瘟菌无毒基因研究进展

稻瘟菌是引起水稻稻瘟病的病原物。水稻与稻瘟菌间存在广泛而特异的相互作用,是研究寄主与病原物互作的重要模式系统。本文对稻瘟菌与水稻互作最重要的激发子―无毒基因的研究现状进行了概括,讨论了无毒基因的定位、克隆方法以及已克隆无毒基因的功能及进化研究,同时对今后无毒基因研究的重要方向进行了探讨,为深入理解无毒基因的功能及与水稻可能的互作关系奠定了基础。     

Organization of chromosome ends in the rice blast fungus, Magnaporthe oryzae

Eukaryotic pathogens of humans often evade the immune system by switching the expression of surface proteins encoded by subtelomeric gene families. To determine if plant pathogenic fungi use a similar mechanism to avoid host defenses, we sequenced the 14 chromosome ends of the rice blast pathogen, Magnaporthe oryzae. One telomere is directly joined to ribosomal RNA-encoding genes, at the end of the ~2 Mb rDNA array. Two are attached to chromosome-unique sequences, and the remainder adjoin a distinct subtelomere region, consisting of a telomere-linked RecQ-helicase (TLH) gene flanked by several blocks of tandem repeats. Unlike other microbes, M.oryzae exhibits very little gene amplification in the subtelomere regions—out of 261 predicted genes found within 100 kb of the telomeres, only four were present at more than one chromosome end. Therefore, it seems unlikely that M.oryzae uses switching mechanisms to evade host defenses. Instead, the M.oryzae telomeres have undergone frequent terminal truncation, and there is evidence of extensive ectopic recombination among transposons in these regions. We propose that the M.oryzae chromosome termini play more subtle roles in host adaptation by promoting the loss of terminally-positioned genes that tend to trigger host defenses.     

MoSNF1 regulates sporulation and pathogenicity in the rice blast fungus Magnaporthe oryzae

The protein kinase Snf1 is a major component of the glucose derepression pathway in yeast and a regulator of gene expression for the cell wall degrading enzyme (CWDE) in some plant pathogenic fungi. To address the molecular function of Snf1 in Magnaporthe oryzae, which causes the rice blast disease, MoSNF1 was cloned and functionally characterized using gene knock-out strategies. MoSNF1 functionally complemented the growth defect of the yeast snf1 mutant on a non-fermenting carbon source. However, the growth rate of the Δmosnf1 mutant on various carbon sources was reduced independent of glucose, and the expression of the CWDE genes in the mutant was induced during derepressing condition like the wild type. The pre-penetration stage including conidial germination and appressorium formation of the Δmosnf1 was largely impaired, and the pathogenicity of the Δmosnf1 was significantly reduced. Most strikingly, the Δmosnf1 mutant produced only a few conidia and had a high frequency of abnormally shaped conidia compared to the wild type. Our results suggest that MoSNF1 is a functional homolog of yeast Snf1, but its contribution to sporulation, vegetative growth and pathogenicity is critical in M. oryzae.     

Light regulation of asexual development in the rice blast fungus, Magnaporthe oryzae

Light is a major environmental factor that influences many biological processes. We characterized the roles of light in asexual development (including the formation of aerial hyphae and conidiophore) in Magnaporthe oryzae, which is the causal agent of rice blast disease. Our data revealed a complex nature of light regulation in the asexual developments of M. oryzae. Asexual development of M. oryzae is suppressed by blue light in a light/dark cycling environment and asexual spore release is controlled by both blue and red light. We demonstrated that even very dim light, about 10 micromol m(-2), is sufficient to suppress spore-release behavior in M. oryzae. We also generated knockout strains of a blue light receptor, mgwc-1, the M. oryzae homolog of white collar-1 in Neurospora crassa, and demonstrated blue-light-specific regulation in the asexual development and spore release in M. oryzae. Our findings in this agriculturally important pathogen, M. oryzae, broaden our understanding of the roles of light in fungal development.     

Infection-associated nuclear degeneration in the rice blast fungus Magnaporthe oryzae requires non-selective macro-autophagy

Background

The rice blast fungus Magnaporthe oryzae elaborates a specialized infection structure called an appressorium to breach the rice leaf surface and gain access to plant tissue. Appressorium development is controlled by cell cycle progression, and a single round of nuclear division occurs prior to appressorium formation. Mitosis is always followed by programmed cell death of the spore from which the appressorium develops. Nuclear degeneration in the spore is known to be essential for plant infection, but the precise mechanism by which it occurs is not known.

Methodology/Principal Findings

In yeast, nuclear breakdown requires a specific form of autophagy, known as piecemeal microautophagy of the nucleus (PMN), and we therefore investigated whether this process occurs in the rice blast fungus. Here, we report that M. oryzae possesses two conserved components of a putative PMN pathway, MoVac8 and MoTsc13, but that both are dispensable for nuclear breakdown during plant infection. MoVAC8 encodes a vacuolar membrane protein and MoTSC13 a peri-nuclear and peripheral ER protein.

Conclusions/Significance

We show that MoVAC8 is necessary for caffeine resistance, but dispensable for pathogenicity of M. oryzae, while MoTSC13 is involved in cell wall stress responses and is an important virulence determinant. By functional analysis of ΔMoatg1 and ΔMoatg4 mutants, we demonstrate that infection-associated nuclear degeneration in M. oryzae instead occurs by non-selective macroautophagy, which is necessary for rice blast disease.     

Mapping of avirulence genes in the rice blast fungus, Magnaporthe grisea, with RFLP and RAPD markers

Three genetically independent avirulence genes, AVR1-Irat7, AVRI-MedNoi; and AVR1-Ku86, were identified in a cross involving isolates Guy11 and 2/0/3 of the rice blast fungus, Magnaporthe grisea. Using 76 random progeny, we constructed a partial genetic map with restriction fragment length polymorphism (RFLP) markers revealed by probes such as the repeated sequences MGL/MGR583 and Pot3/MGR586, cosmids from the M. grisea genetic map, and a telomere sequence oligonucleotide. Avirulence genes AVR1-MedNoi and AVR1-Ku86 were closely linked to telomere RFLPs such as marker TelG (6 cM from AVR1-MedNoi) and TelF (4.5 cM from AVR1-Ku86). Avirulence gene AVR1-Irat7 was linked to a cosmid RFLP located on chromosome 1 and mapped at 20 cM from the avirulence gene AVR1-CO39. Using bulked segregant analysis, we identified 11 random amplified polymorphic DNA (RAPD) markers closely linked (0 to 10 cM) to the avirulence genes segregating in this cross. Most of these RAPD markers corresponded to junction fragments between known or new transposons and a single-copy sequence. Such junctions or the whole sequences of single-copy RAPD markers were frequently absent in one parental isolate. Single-copy sequences from RAPD markers tightly linked to avirulence genes will be used for positional cloning.     

Characterization of infected process and primary mechanism in rice Acuce defense against rice blast fungus,Magnaporthe oryzae

Plant Molecular Biology - Identification of infection process and defense response during M. oryzae infecting Acuce. Magnaporthe oryzae is a destructive rice pathogen. Recent studies have focused...     

Telomere-targeted retrotransposons in the rice blast fungus Magnaporthe oryzae: agents of telomere instability

The fungus Magnaporthe oryzae is a serious pathogen of rice and other grasses. Telomeric restriction fragments in Magnaporthe isolates that infect perennial ryegrass (prg) are hotspots for genomic rearrangement and undergo frequent, spontaneous alterations during fungal culture. The telomeres of rice-infecting isolates are very stable by comparison. Sequencing of chromosome ends from a number of prg-infecting isolates revealed two related non-LTR retrotransposons (M. oryzae Telomeric Retrotransposons or MoTeRs) inserted in the telomere repeats. This contrasts with rice pathogen telomeres that are uninterrupted by other sequences. Genetic evidence indicates that the MoTeR elements are responsible for the observed instability. MoTeRs represent a new family of telomere-targeted transposons whose members are found exclusively in fungi.     

Genome-wide association mapping of virulence gene in rice blast fungus Magnaporthe oryzae using a genotyping by sequencing approach

Magnaporthe oryzae is a fungal pathogen causing blast disease in many plant species. In this study, seventy three isolates of M. oryzae collected from rice (Oryza sativa) in 1996–2014 were genotyped using a genotyping-by-sequencing approach to detect genetic variation. An association study was performed to identify single nucleotide polymorphisms (SNPs) associated with virulence genes using 831 selected SNP and infection phenotypes on local and improved rice varieties. Population structure analysis revealed eight subpopulations. The division into eight groups was not related to the degree of virulence. Association mapping showed five SNPs associated with fungal virulence on chromosome 1, 2, 3, 4 and 7. The SNP on chromosome 1 was associated with virulence against RD6-Pi7 and IRBL7-M which might be linked to the previously reported AvrPi7.     

Regulation of the MPG1 hydrophobin gene in the rice blast fungus Magnaporthe grisea

The hydrophobin-encoding gene MPG1 of the rice blast fungus Magnaporthe grisea is highly expressed during the initial stages of host plant infection and targeted deletion of the gene results in a mutant strain that is reduced in virulence, conidiation, and appressorium formation. The green fluorescent protein-encoding allele sGFP was used as a reporter to investigate regulatory genes that control MPG1 expression. The MAP kinase-encoding gene PMK1 and the wide domain regulators of nitrogen source utilization, NPR1 and NUT1, were required for full expression of MPG1 in response to starvation stress. The CPKA gene, encoding the catalytic subunit of protein kinase A, was required for repression of MPG1 during growth in rich nutrient conditions. During appressorium morphogenesis, high-level MPG1 expression was found to require the CPKA and NPR1 genes. Expression of a destabilized GFP allele indicated that de novo MPG1 expression occurs during appressorium formation. Three regions of the MPG1 promoter were identified which are required for high-level expression of MPG1 during appressorium formation and are necessary for the biological activity of the MPG1 hydrophobin during spore formation and plant infection.     

湖南桃江病圃稻瘟病菌的无毒基因及水稻抗瘟单基因联合抗性分析

【目的】鉴定湖南省桃江病圃稻瘟病菌无毒基因型,为合理搭配种植湖南省水稻抗瘟品种和抗病育种提供依据。【方法】在湖南桃江病圃采集水稻品种"丽江新团黑谷"(LTH)稻瘟菌病样,用单孢分离法分离稻瘟病菌单孢并纯化获得单孢菌株,用针刺离体法将菌株接种到以"LTH"为轮回亲本培育而成的24个含单抗瘟基因的水稻5叶期第5叶片上,对供试菌株进行无毒基因鉴定,并应用联合致病性系数和联合抗病性系数分析抗瘟基因组合间的互作。【结果】供试92个稻瘟病单孢菌株含有全部的24个无毒基因,对24个已知含单抗瘟基因的水稻材料表现出不同程度的毒力水平,含水稻抗瘟基因Pi-20对供试菌株抗菌频率最高,达54.35%;通过联合致病性系数和联合抗病性系数分析抗瘟基因组合间的互作,结果表明最佳搭配组合为Pi-20×Pi-k~s(RAC=0.28,PAC=0.23)。【结论】湖南省桃江病圃稻瘟病菌致病力较强,24个抗瘟基因多已感病化,含抗性基因Pi-20与Pi-k、Pi-k~s、Pi-3组合的水稻品种目前可在湖南省推广利用,但需研究引进新的抗瘟基因。