Genome analyses reveal the hybrid origin of the staple crop white Guinea yam (Dioscorea rotundata)

西アフリカの主食作物ギニアヤムの起源を解明 --ギニアヤムはサバンナと熱帯雨林に生育する野生種の雑種起源--. 京都大学プレスリリース. 2020-12-11.White Guinea yam (Dioscorea rotundata) is an important staple tuber crop in West Africa. However, its origin remains unclear. In this study, we resequenced 336 accessions of white Guinea yam and compared them with the sequences of wild Dioscorea species using an improved reference genome sequence of D. rotundata. In contrast to a previous study suggesting that D. rotundata originated from a subgroup of Dioscorea praehensilis, our results suggest a hybrid origin of white Guinea yam from crosses between the wild rainforest species D. praehensilis and the savannah-adapted species Dioscorea abyssinica. We identified a greater genomic contribution from D. abyssinica in the sex chromosome of Guinea yam and extensive introgression around the SWEETIE gene. Our findings point to a complex domestication scenario for Guinea yam and highlight the importance of wild species as gene donors for improving this crop through molecular breeding

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

A genetically linked pair of NLR immune receptors shows contrasting patterns of evolution

イネのいもち病抵抗性機構の解明 --イネ抵抗性タンパク質の付加ドメインが擬似餌となり多様な病原菌因子を釣り上げて見破る--. 京都大学プレスリリース. 2022-07-22.Throughout their evolution, plant nucleotide-binding leucine-rich-repeat receptors (NLRs) have acquired widely divergent unconventional integrated domains that enhance their ability to detect pathogen effectors. However, the functional dynamics that drive the evolution of NLRs with integrated domains (NLR-IDs) remain poorly understood. Here, we reconstructed the evolutionary history of an NLR locus prone to unconventional domain integration and experimentally tested hypotheses about the evolution of NLR-IDs. We show that the rice (Oryza sativa) NLR Pias recognizes the effector AVR-Pias of the blast fungal pathogen Magnaporthe oryzae. Pias consists of a functionally specialized NLR pair, the helper Pias-1 and the sensor Pias-2, that is allelic to the previously characterized Pia pair of NLRs: the helper RGA4 and the sensor RGA5. Remarkably, Pias-2 carries a C-terminal DUF761 domain at a similar position to the heavy metal-associated (HMA) domain of RGA5. Phylogenomic analysis showed that Pias-2/RGA5 sensor NLRs have undergone recurrent genomic recombination within the genus Oryza, resulting in up to six sequence-divergent domain integrations. Allelic NLRs with divergent functions have been maintained transspecies in different Oryza lineages to detect sequence-divergent pathogen effectors. By contrast, Pias-1 has retained its NLR helper activity throughout evolution and is capable of functioning together with the divergent sensor-NLR RGA5 to respond to AVR-Pia. These results suggest that opposite selective forces have driven the evolution of paired NLRs: highly dynamic domain integration events maintained by balancing selection for sensor NLRs, in sharp contrast to purifying selection and functional conservation of immune signaling for helper NLRs

<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

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

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

Microscopic analysis suggests that <i>MC69</i> promoter is constitutively active and MC69::mCherry fusion protein is secreted.

<p>(A) and (D) Schematic diagrams of mCherry and MC69::mCherry fusion protein expression constructs. (B) and (E) Conidia from WT+<i>mCherry</i> and <i>mc69</i>+<i>MC69::mCherry</i> were harvested, and appressorium development was observed for 12 h on glass coverslips. Merged DIC and mCherry (red) images were taken. Scale bars = 10 µm. (C) and (F) Merged DIC and mCherry images of the rice leaf sheath cells infected with mCherry- and MC69::mCherry-expressing transformants 24 h and 48 h after inoculation. Scale bars = 20 µm. (G) Blast symptoms caused by Ina72 WT, <i>mc69</i> and <i>mc69</i>+<i>MC69::Cherrry</i> on rice (cv. Shin No. 2) 7 days after inoculation.</p