The allelic rice immune receptor Pikh confers extended resistance to strains of the blast fungus through a single polymorphism in the effector binding interface

Arms race co-evolution drives rapid adaptive changes in pathogens and in the immune systems of their hosts. Plant intracellular NLR immune receptors detect effectors delivered by pathogens to promote susceptibility, activating an immune response that halts colonization. As a consequence, pathogen effectors evolve to escape immune recognition and are highly variable. In turn, NLR receptors are one of the most diverse protein families in plants, and this variability underpins differential recognition of effector variants. The molecular mechanisms underlying natural variation in effector recognition by NLRs are starting to be elucidated. The rice NLR pair Pik-1/Pik-2 recognizes AVR-Pik effectors from the blast fungus Magnaporthe oryzae, triggering immune responses that limit rice blast infection. Allelic variation in a heavy metal associated (HMA) domain integrated in the receptor Pik-1 confers differential binding to AVR-Pik variants, determining resistance specificity. Previous mechanistic studies uncovered how a Pik allele, Pikm, has extended recognition to effector variants through a specialized HMA/AVR-Pik binding interface. Here, we reveal the mechanistic basis of extended recognition specificity conferred by another Pik allele, Pikh. A single residue in Pikh-HMA increases binding to AVR-Pik variants, leading to an extended effector response in planta. The crystal structure of Pikh-HMA in complex with an AVR-Pik variant confirmed that Pikh and Pikm use a similar molecular mechanism to extend their pathogen recognition profile. This study shows how different NLR receptor alleles functionally converge to extend recognition specificity to pathogen effectors.     

Association Genetics Reveals Three Novel Avirulence Genes from the Rice Blast Fungal Pathogen Magnaporthe oryzae

To subvert rice (Oryza sativa) host defenses, the devastating ascomycete fungus pathogen Magnaporthe oryzae produces a battery of effector molecules, including some with avirulence (AVR) activity, which are recognized by host resistance (R) proteins resulting in rapid and effective activation of innate immunity. To isolate novel avirulence genes from M. oryzae, we examined DNA polymorphisms of secreted protein genes predicted from the genome sequence of isolate 70-15 and looked for an association with AVR activity. This large-scale study found significantly more presence/absence polymorphisms than nucleotide polymorphisms among 1032 putative secreted protein genes. Nucleotide diversity of M. oryzae among 46 isolates of a worldwide collection was extremely low (θ = 8.2 × 10⁻⁵), suggestive of recent pathogen dispersal. However, no association between DNA polymorphism and AVR was identified. Therefore, we used genome resequencing of Ina168, an M. oryzae isolate that contains nine AVR genes. Remarkably, a total of 1.68 Mb regions, comprising 316 candidate effector genes, were present in Ina168 but absent in the assembled sequence of isolate 70-15. Association analyses of these 316 genes revealed three novel AVR genes, AVR-Pia, AVR-Pii, and AVR-Pik/km/kp, corresponding to five previously known AVR genes, whose products are recognized inside rice cells possessing the cognate R genes. AVR-Pia and AVR-Pii have evolved by gene gain/loss processes, whereas AVR-Pik/km/kp has evolved by nucleotide substitutions and gene gain/loss.     

Genomic rearrangements generate hypervariable mini-chromosomes in host-specific isolates of the blast fungus

Supernumerary mini-chromosomes–a unique type of genomic structural variation–have been implicated in the emergence of virulence traits in plant pathogenic fungi. However, the mechanisms that facilitate the emergence and maintenance of mini-chromosomes across fungi remain poorly understood. In the blast fungus Magnaporthe oryzae (Syn. Pyricularia oryzae), mini-chromosomes have been first described in the early 1990s but, until very recently, have been overlooked in genomic studies. Here we investigated structural variation in four isolates of the blast fungus M. oryzae from different grass hosts and analyzed the sequences of mini-chromosomes in the rice, foxtail millet and goosegrass isolates. The mini-chromosomes of these isolates turned out to be highly diverse with distinct sequence composition. They are enriched in repetitive elements and have lower gene density than core-chromosomes. We identified several virulence-related genes in the mini-chromosome of the rice isolate, including the virulence-related polyketide synthase Ace1 and two variants of the effector gene AVR-Pik. Macrosynteny analyses around these loci revealed structural rearrangements, including inter-chromosomal translocations between core- and mini-chromosomes. Our findings provide evidence that mini-chromosomes emerge from structural rearrangements and segmental duplication of core-chromosomes and might contribute to adaptive evolution of the blast fungus.     

Backbone and sidechain <Superscript>1</Superscript>H, <Superscript>13</Superscript>C and <Superscript>15</Superscript>N chemical shift assignments of the hydrophobin MPG1 from the rice blast fungus <Emphasis Type="Italic">Magnaporthe oryzae</Emphasis>

Fungal hydrophobins are secreted proteins that self-assemble at hydrophobic:hydrophilic interfaces. They are essential for a variety of processes in the fungal life cycle, including mediating interactions with surfaces and infection of hosts. The fungus Magnaporthe oryzae, the causative agent of rice blast, relies on the unique properties of hydrophobins to infect cultivated rice as well as over 50 different grass species. The hydrophobin MPG1 is highly expressed during rice blast pathogenesis and has been implicated during host infection. Here we report the backbone and sidechain assignments for the class I hydrophobin MPG1 from the rice blast fungus Magnaporthe oryzae.     

Variability in Aggressiveness of Rice Blast (Magnaporthe oryzae) Isolates Originating from Rice Leaves and Necks: A Case of Pathogen Specialization?

Rice blast, caused by Magnaporthe oryzae, causes yield losses associated with injuries on leaves and necks, the latter being in general far more important than the former. Many questions remain on the relationships between leaf and neck blast, including questions related to the population biology of the pathogen. Our objective was to test the hypothesis of adaptation of M. oryzae isolates to the type of organ they infect. To that aim, the components of aggressiveness of isolates originating from leaves and necks were measured. Infection efficiency, latent period, sporulation intensity, and lesion size were measured on both leaves and necks. Univariate and multivariate analyses indicated that isolates originating from leaves were less aggressive than isolates originating from necks, when aggressiveness components were measured on leaves as well as on necks, indicating that there is no specialization within the pathogen population with respect to the type of organ infected. This result suggests that the more aggressive isolates involved in epidemics on leaves during the vegetative stage of the crop cycle have a higher probability to infect necks, and that a population shift may occur during disease transmission from leaves to necks. Implications for disease management are discussed.     

Overexpression of a Novel Biotrophy-Specific Colletotrichum truncatum Effector,CtNUDIX, in Hemibiotrophic Fungal Phytopathogens Causes Incompatibility with Their Host Plants

The hemibiotrophic fungus Colletotrichum truncatum causes anthracnose disease on lentils and a few other grain legumes. It shows initial symptomless intracellular growth, where colonized host cells remain viable (biotrophy), and then switches to necrotrophic growth, killing the colonized host plant tissues. Here, we report a novel effector gene, CtNUDIX, from C. truncatum that is exclusively expressed during the late biotrophic phase (before the switch to necrotrophy) and elicits a hypersensitive response (HR)-like cell death in tobacco leaves transiently expressing the effector. CtNUDIX homologs, which contain a signal peptide and a Nudix hydrolase domain, may be unique to hemibiotrophic fungal and fungus-like plant pathogens. CtNUDIX lacking a signal peptide or a Nudix motif failed to induce cell death in tobacco. Expression of CtNUDIX:eGFP in tobacco suggested that the fusion protein might act on the host cell plasma membrane. Overexpression of CtNUDIX in C. truncatum and the rice blast pathogen, Magnaporthe oryzae, resulted in incompatibility with the hosts lentil and barley, respectively, by causing an HR-like response in infected host cells associated with the biotrophic invasive hyphae. These results suggest that C. truncatum and possibly M. oryzae elicit cell death to signal the transition from biotrophy to necrotrophy.     

Cells in cells: morphogenetic and metabolic strategies conditioning rice infection by the blast fungus Magnaporthe oryzae

The rice blast fungus Magnaporthe oryzae is a global food security threat due to its destruction of cultivated rice. Of the world's rice harvest, 10–30 % is lost each year to this pathogen, and changing climates are likely to favor its spread into new areas. Insights into how the fungus might be contained could come from the wealth of molecular and cellular studies that have been undertaken in order to shed light on the biological underpinnings of blast disease, aspects of which we review herein. Infection begins when a three-celled spore lands on the surface of a leaf, germinates, and develops the specialized infection structure called the appressorium. The mature appressorium develops a high internal turgor that acts on a thin penetration peg, forcing it through the rice cuticle and into the underlying epidermal cells. Primary then invasive hyphae (IH) elaborate from the peg and grow asymptomatically from one living rice cell to another for the first few days of infection before host cells begin to die and characteristic necrotic lesions form on the surface of the leaf, from which spores are produced to continue the life cycle. To gain new insights into the biology of rice blast disease, we argue that, conceptually, the infection process can be viewed as two discrete phases occurring in markedly different environments and requiring distinct biochemical pathways and morphogenetic regulation: outside the host cell, where the appressorium develops in a nutrient-free environment, and inside the host cell, where filamentous growth occurs in a glucose-rich, nitrogen-poor environment, at least from the perspective of the fungus. Here, we review the physiological and metabolic changes that occur in M. oryzae as it transitions from the surface to the interior of the host, thus enabling us to draw lessons about the strategies that allow M. oryzae cells to thrive in rice cells.     

The evolution of generalization? Parasitoid flies and the perils of inferring host range evolution from phylogenies

It is widely assumed that high resource specificity predisposes lineages toward greater likelihood of extinction and lower likelihood of diversification than more generalized lineages. This suggests that host range evolution in parasitic organisms should proceed from generalist to specialist, and specialist lineages should be found at the 'tips' of phylogenies. To test these hypotheses, parsimony and maximum likelihood methods were used to reconstruct the evolution of host range on a phylogeny of parasitoid flies in the family Tachinidae. In contrast to predictions, most reconstructions indicated that generalists were repeatedly derived from specialist lineages and tended to occupy terminal branches of the phylogeny. These results are critically examined with respect to hypotheses concerning the evolution of specialization, the inherent difficulties in inferring host ranges, our knowledge of tachinid-host associations, and the methodological problems associated with ancestral character state reconstruction. Both parsimony and likelihood reconstructions are shown to provide misleading results and it is argued that independent evidence, in addition to phylogenetic trees, is needed to inform models of the evolution of host range and the evolutionary consequences of specialization.     

Sequence variation of avirulence gene AVR-Pita1 in rice blast fungus, Magnaporthe oryzae

The interaction between rice, Oryza sativa, and rice blast fungus, Magnaporthe oryzae, is triggered by an interaction between the protein products of the host resistant gene, and the pathogen avirulence gene. This interaction follows the ‘gene-for-gene' concept. The resistant gene has effectively protected rice plants from rice blast infection. However, the resistant genes usually break down several years after the release of the resistant rice varieties because the fungus has evolved to new races. The objective of this study is to investigate the nucleotide sequence variation of the AVR-Pita1 gene that influences the adaption of rice blast fungus to overcome the resistant gene, Pi-ta. Thirty rice blast fungus isolates were collected in 2005 and 2010 from infected rice plants in northern and northeastern Thailand. The nucleotide sequences of AVR-Pita1 were amplified and analyzed. Phylogenetic analysis was conducted using the MEGA 5.0 program. The results showed a high level of nucleotide sequence polymorphisms and the positive genetic selection pressure in Thai rice blast isolates. The details of sequence variation analysis were described in this article. The information from this study can be used for rice blast resistant breeding program in the future.     

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.     

A nonclassically secreted effector of Magnaporthe oryzae targets host nuclei and plays important roles in fungal growth and plant infection

Rice blast caused by Magnaporthe oryzae is one of the most destructive diseases and poses a growing threat to food security worldwide. Like many other filamentous pathogens, rice blast fungus releases multiple types of effector proteins to facilitate fungal infection and modulate host defence responses. However, most of the characterized effectors contain an N-terminal signal peptide. Here, we report the results of the functional characterization of a nonclassically secreted nuclear targeting effector in M. oryzae (MoNte1). MoNte1 has no signal peptide, but can be secreted and translocated into plant nuclei driven by a nuclear targeting peptide. It could also induce hypersensitive cell death when transiently expressed in Nicotiana benthamiana. Deletion of the MoNTE1 gene caused a significant reduction of fungal growth and conidiogenesis, partially impaired appressorium formation and host colonization, and also dramatically attenuated the pathogenicity. Taken together, these findings reveal a novel effector secretion pathway and deepen our understanding of rice–M. oryzae interactions.     

Rice blast fungus (Magnaporthe oryzae) infects Arabidopsis via a mechanism distinct from that required for the infection of rice

Magnaporthe oryzae is a hemibiotrophic fungal pathogen that causes rice (Oryza sativa) blast. Although M. oryzae as a whole infects a wide variety of monocotyledonous hosts, no dicotyledonous plant has been reported as a host. We found that two rice pathogenic strains of M. oryzae, KJ201 and 70-15, interacted differentially with 16 ecotypes of Arabidopsis (Arabidopsis thaliana). Strain KJ201 infected all ecotypes with varying degrees of virulence, whereas strain 70-15 caused no symptoms in certain ecotypes. In highly susceptible ecotypes, small chlorotic lesions appeared on infected leaves within 3 d after inoculation and subsequently expanded across the affected leaves. The fungus produced spores in susceptible ecotypes but not in resistant ecotypes. Fungal cultures recovered from necrotic lesions caused the same symptoms in healthy plants, satisfying Koch's postulates. Histochemical analyses showed that infection by the fungus caused an accumulation of reactive oxygen species and eventual cell death. Similar to the infection process in rice, the fungus differentiated to form appressorium and directly penetrated the leaf surface in Arabidopsis. However, the pathogenic mechanism in Arabidopsis appears distinct from that in rice; three fungal genes essential for pathogenicity in rice played only limited roles in causing disease symptoms in Arabidopsis, and the fungus seems to colonize Arabidopsis as a necrotroph through the secretion of phytotoxic compounds, including 9,12-octadecadienoic acid. Expression of PR-1 and PDF1.2 was induced in response to infection by the fungus, suggesting the activation of salicylic acid- and jasmonic acid/ethylene-dependent signaling pathways. However, the roles of these signaling pathways in defense against M. oryzae remain unclear. In combination with the wealth of genetic and genomic resources available for M. oryzae, this newly established pathosystem allows comparison of the molecular and cellular mechanisms underlying pathogenesis and host defense in two well-studied model plants.     

The biotrophy-associated secreted protein 4 (BAS4) participates in the transition of Magnaporthe oryzae from the biotrophic to the necrotrophic phase

The physiological and metabolic processes of host plants are manipulated and remodeled by phytopathogenic fungi during infection, revealed obvious signs of biotrophy of the hemibiotrophic pathogen. As we known that effector proteins play key roles in interaction of hemibiotrophic fungi and their host plants. BAS4 (biotrophy-associated secreted protein 4) is an EIHM (extrainvasive hyphal membrane) matrix protein that was highly expressed in infectious hyphae. In order to study whether BAS4 is involved in the transition of rice blast fungus from biotrophic to necrotrophic phase, The susceptible rice cultivar Lijiangxintuanheigu (LTH) that were pre-treated with prokaryotic expression product of BAS4 and then followed with inoculation of the blast strain, more serious blast disease symptom, more biomass such as sporulation and fungal relative growth, and lower expression level of pathogenicity-related genes appeared in lesion of the rice leaves than those of the PBS-pretreated-leaves followed with inoculation of the same blast strain, which demonstrating that BAS4 invitro changed rice defense system to facilitate infection of rice blast strain. And the susceptible rice cultivar (LTH) were inoculated withBAS4-overexpressed blast strain, we also found more serious blast disease symptom and more biomass also appeared in lesion of leaves inoculated with BAS4-overexpressed strain than those of leaves inoculated with the wild-type strain, and expression level of pathogenicity-related genes appeared lower in biotrophic phase and higher in necrotrophic phase of infection, indicating BAS4 maybe in vivo regulate defense system of rice to facilitate transition of biotrophic to necrotrophic phase. Our data demonstrates that BAS4 in vitro and in vivo participates in transition from the biotrophic to the necrotrophic phase of Magnaporthe oryzae.     

Relationship between Phylogeny and Pathotype for the Bacterial Blight Pathogen of Rice

Several transposable elements were isolated from the genome of Xanthomonas oryzae pv. oryzae. These elements and an avirulence gene isolated from X. oryzae pv. oryzae were used as hybridization probes for a collection of X. oryzae pv. oryzae strains from the Philippines. Each of the sequences was present in multiple copies in all strains examined and showed distinct patterns of hybridizing bands. Phenograms were derived from the restriction fragment length polymorphism data obtained for each of the individual probes and for pooled data from multiple probes. The phenograms derived from the different probes differed in topology and, on the basis of bootstrap analysis, were not equally robust. For all of the probes, including the avirulence gene, some groups (even some haplotypes) consisted of multiple races. The strains were grouped into four major clusters on the basis of the two probes giving the highest bootstrap values. These groups were inferred to represent phylogenetic lineages. Three of the six races of X. oryzae pv. oryzae appeared in more than one of the lineages, and another was present in two sublineages. For three of the races, strains representing different phenetic groups were inoculated on rice cultivars carrying 10 resistance genes. Two new races were differentiated, corresponding to pathogen lineages identified by DNA typing. On the basis of DNA and pathotypic analyses, together with information on the spatial and temporal distribution of the pathogen types from this and other studies, a general picture of X. oryzae pv. oryzae evolution in the Philippines is presented.     

Toward understanding of rice innate immunity against Magnaporthe oryzae

The blast fungus, Magnaporthe oryzae, causes serious disease on a wide variety of grasses including rice, wheat and barley. The recognition of pathogens is an amazing ability of plants including strategies for displacing virulence effectors through the adaption of both conserved and variable pathogen elicitors. The pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI) and effector-triggered immunity (ETI) were reported as two main innate immune responses in plants, where PTI gives basal resistance and ETI confers durable resistance. The PTI consists of extracellular surface receptors that are able to recognize PAMPs. PAMPs detect microbial features such as fungal chitin that complete a vital function during the organism’s life. In contrast, ETI is mediated by intracellular receptor molecules containing nucleotide-binding (NB) and leucine rich repeat (LRR) domains that specifically recognize effector proteins produced by the pathogen. To enhance crop resistance, understanding the host resistance mechanisms against pathogen infection strategies and having a deeper knowledge of innate immunity system are essential. This review summarizes the recent advances on the molecular mechanism of innate immunity systems of rice against M. oryzae. The discussion will be centered on the latest success reported in plant–pathogen interactions and integrated defense responses in rice.