Cross-reactivity of a rice NLR immune receptor to distinct effectors from the rice blast pathogen Magnaporthe oryzae provides partial disease resistance

Unconventional integrated domains in plant intracellular immune receptors of the nucleotide-binding leucine-rich repeat (NLRs) type can directly bind translocated effector proteins from pathogens and thereby initiate an immune response. The rice (Oryza sativa) immune receptor pairs Pik-1/Pik-2 and RGA5/RGA4 both use integrated heavy metal-associated (HMA) domains to bind the effectors AVR-Pik and AVR-Pia, respectively, from the rice blast fungal pathogen Magnaporthe oryzae These effectors both belong to the MAX effector family and share a core structural fold, despite being divergent in sequence. How integrated domains in NLRs maintain specificity of effector recognition, even of structurally similar effectors, has implications for understanding plant immune receptor evolution and function. Here, using plant cell death and pathogenicity assays and protein-protein interaction analyses, we show that the rice NLR pair Pikp-1/Pikp-2 triggers an immune response leading to partial disease resistance toward the "mis-matched" effector AVR-Pia in planta and that the Pikp-HMA domain binds AVR-Pia in vitro We observed that the HMA domain from another Pik-1 allele, Pikm, cannot bind AVR-Pia, and it does not trigger a plant response. The crystal structure of Pikp-HMA bound to AVR-Pia at 1.9 Å resolution revealed a binding interface different from those formed with AVR-Pik effectors, suggesting plasticity in integrated domain-effector interactions. The results of our work indicate that a single NLR immune receptor can bait multiple pathogen effectors via an integrated domain, insights that may enable engineering plant immune receptors with extended disease resistance profiles

Large-Scale Gene Disruption in Magnaporthe oryzae Identifies MC69, a Secreted Protein Required for Infection by Monocot and Dicot Fungal Pathogens

To search for virulence effector genes of the rice blast fungus, Magnaporthe oryzae, we carried out a large-scale targeted disruption of genes for 78 putative secreted proteins that are expressed during the early stages of infection of M. oryzae. Disruption of the majority of genes did not affect growth, conidiation, or pathogenicity of M. oryzae. One exception was the gene MC69. The mc69 mutant showed a severe reduction in blast symptoms on rice and barley, indicating the importance of MC69 for pathogenicity of M. oryzae. The mc69 mutant did not exhibit changes in saprophytic growth and conidiation. Microscopic analysis of infection behavior in the mc69 mutant revealed that MC69 is dispensable for appressorium formation. However, mc69 mutant failed to develop invasive hyphae after appressorium formation in rice leaf sheath, indicating a critical role of MC69 in interaction with host plants. MC69 encodes a hypothetical 54 amino acids protein with a signal peptide. Live-cell imaging suggested that fluorescently labeled MC69 was not translocated into rice cytoplasm. Site-directed mutagenesis of two conserved cysteine residues (Cys36 and Cys46) in the mature MC69 impaired function of MC69 without affecting its secretion, suggesting the importance of the disulfide bond in MC69 pathogenicity function. Furthermore, deletion of the MC69 orthologous gene reduced pathogenicity of the cucumber anthracnose fungus Colletotrichum orbiculare on both cucumber and Nicotiana benthamiana leaves. We conclude that MC69 is a secreted pathogenicity protein commonly required for infection of two different plant pathogenic fungi, M. oryzae and C. orbiculare pathogenic on monocot and dicot plants, respectively

植物認識によって活性化されるウリ類炭疽病菌の感染機構に関与する遺伝子の研究

京都大学0048新制・課程博士博士(農学)甲第17266号農博第1966号新制||農||1007(附属図書館)学位論文||H25||N4724(農学部図書室)30023京都大学大学院農学研究科応用生物科学専攻(主査)教授 奥野 哲郎, 教授 佐久間 正幸, 教授 田中 千尋学位規則第4条第1項該当Doctor of Agricultural ScienceKyoto UniversityDA

Septin-mediated plant cell invasion by the rice blast fungus, Magnaporthe oryzae

Blasting Through The fungus that causes rice blast disease, Magnaporthe oryzae , can lead to devastating reductions in rice yields. M. oryzae enters the plant by developing specialized infection structures called appressoria. Appressoria generate enormous internal turgor pressure that somehow creates invasive forces that physically break the plant cuticle. Dagdas et al. (p. 1590 ) found that a toroidal (doughnut-shaped) filamentous actin network forms at the base of the appressorium at the precise point where the penetration peg, which ruptures the rice leaf cuticle, will emerge. This network is scaffolded by means of four septin guanosine triphosphatases, which form a dynamic ring structure that colocalizes with F-actin. The findings reveal how fungi translate extreme pressure into localized physical force. </jats:p

Secretion of MC69(C36A)::mCherry and MC69::mCherry fusion proteins.

<p>(A) Schematic diagram of MC69(36A)::mCherry fusion protein expression construct. (B) Merged DIC and mCherry images of rice leaf sheath cells infected by the MC69(C36A)::mCherry-expressing transformants 24 h and 48 h after inoculation. Scale bar = 20 µm. (C) Western blot probed with an anti-DsRed antibody. Samples were loaded as follows: lane 1, culture filtrate from mCherry-expressing strain; lane 2, culture filtrate from MC69::mCherry-expressing strain; lane 3, culture filtrate from MC69(C36A)::mCherry-expressing strain.</p

<i>CoMC69</i> is involved in fungal pathogenicity of <i>C. orbiculare</i>.

<p>(A) Sequence alignment of MC69 between <i>M. oryzae</i> (Mo) and <i>C. orbiculare</i> (Co). Amino acid sequences were aligned using Clustal W program <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002711#ppat.1002711-Tompson1" target="_blank">[52]</a>. Identical amino acids are indicated as white letters on a black background. Similar residues are shown by gray background. Gaps introduced for alignment are indicated by hyphens. (B) Pathogenicity test of the <i>Comc69</i> mutants on cucumber. Conidial suspensions were inoculated on detached cotyledons of cucumber (<i>Cucumis sativa</i>). On the left half of the cotyledons, the wild-type strain 104-T was inoculated as positive control. On the right half, the <i>Comc69</i> strains (DMC1 and DMC2) were inoculated. Inoculated cotyledons were incubated for 7 days. (C) Pathogenicity test of the <i>Comc69</i> mutants on <i>N. benthamiana</i>. On the left half of the detached leaves of <i>N. benthamiana</i>, the strain 104-T was inoculated as positive control. On the right half, the <i>Comc69</i> strains (DMC1 and DMC2) were inoculated. Inoculated leaves were incubated for 7 days. (D) mCherry-based reporter assay for expression of the <i>CoMC69</i> gene. Conidia from the <i>C. orbiculare</i> strain carrying the <i>CoMC69</i> promoter-<i>mCherry</i> fusion gene (<i>CoMC69p::mCherry</i>) was inoculated onto the lower surfaces of cucumber cotyledons, and the inoculated plant was incubated for 4 days. a, appressorium; ih, intracellular hypha. Scale bars = 10 µm.</p

<i>MC69</i> is involved in appressorial penetration and pathogenicity of <i>M. oryzae</i>.

<p>(A) <i>MC69</i> is required for pathogenicity of <i>M. oryzae</i> strain Ina72. Conidial suspension of the wild-type strain Ina72 (Ina72 WT) and the <i>mc69</i> mutants (<i>mc69-9</i>, <i>-12</i>, <i>-87</i>) were inoculated on barley (cv. Nigrate) and rice (cv. Shin No. 2) leaves, and incubated for 4 and 7 days, respectively. (B) Germination, appressorium formation and appressorial penetration of Ina72 WT and the <i>mc69</i> mutants. The ratio of germination was calculated as the mean percentage of conidia germinated after 24 h on rice (cv. Shin No. 2) leaf sheath cells. The mean percentage of appressorium formation on rice leaf sheath cells among the germinated conidia is presented. Three replicates of ∼50 conidia were counted for each observation. The mean percentage of appressorial penetration by the <i>mc69</i> mutants 32 h after inoculation is presented. Standard errors are indicated by the vertical bars. (C) Appressorial penetration assays on rice leaf sheath cells. Conidia from Ina72 WT and <i>mc69-87</i> germinated and formed melanized appressoria. All appressoria formed by Ina72 WT penetrated and produced infectious hyphae but no appressoria formed by <i>mc69-87</i> produced infectious hyphae in most of area. Photographs were taken 32 h after inoculation. Scale bar = 40 µm. (D) Invasive growth rating of rice leaf sheath cells 32 h after inoculating with Ina72 WT and <i>mc69-87</i>. The levels for invasive growth rating are given above. For details of the invasive growth levels and rating see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002711#s4" target="_blank">Materials and Methods</a>. Scale bar = 20 µm. (E), (F) Complementation of <i>mc69</i> mutant with the wild type allele of <i>MC69</i>. (E) Appressorial penetration by Ina72 WT, <i>mc69-87</i> (<i>mc69</i>) and the <i>MC69</i> re-introduced strain (<i>mc69</i>+<i>MC69</i>). Mean percentage of appressorial penetration is recorded 32 h after inoculation in rice leaf sheath cells. Four replicates of ∼50 appressoria were counted for each observation. (F) Blast symptoms caused by Ina72 WT, <i>mc69</i> and <i>mc69</i>+<i>MC69</i> on barley (cv. Nigrate) and on rice (cv. Shin No 2) 3 and 4 days after inoculation, respectively.</p

MC69::mCherry is not translocated into the rice cytoplasm.

<p>Merged DIC and mCherry images of rice leaf sheath cells infected by <i>M. oryzae</i> Sasa2 strain harboring (A) <i>PWL2p::PWL2::mCherry::NLS</i>, (B) <i>PWL2p::MC69::mCherry::NLS</i>, and (C) <i>PWL2p::MC69::mCherry</i> 24, 27 and 32 h after inoculation as observed by confocal laser scanning microscopy. Arrows indicate BICs and triangles indicate rice nuclei. Pinhole setting is 240 µm for all panels. Scale bar = 20 µm.</p

<i>MC69</i> is necessary for appressorial penetration and pathogenicity of a laboratory strain 70-15.

<p>(A) <i>MC69</i> is required for pathogenicity of <i>M. oryzae</i> strain 70-15. Conidial suspension of the wild-type strain 70-15 (70-15 WT) and the <i>mc69</i> mutants (<i>mc69-119</i>, <i>-31</i>) were inoculated on barley (cv. Nigrate) and rice (cv. Shin No. 2) leaves, and incubated for 7 days. (B) Germination, appressorium formation and appressorial penetration of 70-15 WT and the <i>mc69</i> mutants. The ratio of germination was calculated as the mean percentage of conidia germinated after 32 h on rice (cv. Shin No. 2) leaf sheath cells. The mean percentage of appressorium formation on rice leaf sheath cells among the germinated conidia is presented. Three replicates of ∼50 conidia were counted for each observation. The mean percentage of appressorial penetration by the <i>mc69</i> mutants is presented 32 h after inoculation. Standard errors are indicated by the vertical bars.</p