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International audience; Several plant pathogenic fungi including Colletotrichum species form specialized cells called appressoria to directly penetrate the intact cuticles of their host plants. These appressoria-forming fungi can infect a wide range of plant species including many commercially important fruits, vegetables, and cereals. The differentiation of appressorium relies on the detection and transduction of physical and biochemical signals present on the plant surface. Working with the cucumber anthracnose fungus Colletotrichum orbiculare, our research has revealed that long chain aliphatic molecules produced through the hydrolysis of plant surface cuticle by conidial cutinase serves as signaling molecules. Following signal detection, the morphogenesis related NDR (nuclear dbf2-related) kinase pathway, and its downstream transcription factor CoMTF4, play a crucial role in the transduction of plant-derived signal involved in appressorium development. In the stage of plant penetration from appressoria, we have also uncovered the recognition of plant surface signals mediated by a pair of peroxidase/copper radical oxidase (CRO) enzymes. The corresponding pair of genes conserve tandem localization in the genome of most Colletotrichum species and the enzyme pair is co-secreted during the penetration stage. The oxidative action of the paired enzymes on plant cuticular derived long-chain alcohols generates signaling molecules (i.e., aldehydes) that potentially functions in a biochemical cascade. In this article, we review our current understanding of fungal-plant communication in adapting to the environmental conditions required for infection-related morphogenesis.

Global food security is endangered by fungal phytopathogens causing devastating crop production losses. Many of these pathogens use specialized appressoria cells to puncture plant cuticles. Here, we unveil a pair of alcohol oxidase–peroxidase enzymes to be essential for pathogenicity. Using Colletotrichum orbiculare, we show that the enzyme pair is cosecreted by the fungus early during plant penetration and that single and double mutants have impaired penetration ability. Molecular modeling, biochemical, and biophysical approaches revealed a fine-tuned interplay between these metalloenzymes, which oxidize plant cuticular long-chain alcohols into aldehydes. We show that the enzyme pair is involved in transcriptional regulation of genes necessary for host penetration. The identification of these infection-specific metalloenzymes opens new avenues on the role of wax-derived compounds and the design of oxidase-specific inhibitors for crop protection. Fungal phytopathogens secrete tandem metalloenzymes that catalyze cuticle oxidation and drive plant cell entry This study was supported by the “Agence Nationale de la Recherche” and by the Natural Sciences and Engineering Research Council of Canada through the ANR-NSERC project “FUNTASTIC” (ANR-17-CE07-0047, STPGP 493781-16). We are grateful to MANE & Fils and the “Association Nationale Recherche Technologie” (ANRT) for funding the Ph.D. fellowship of D.R. (grant no. 2017/1169). Work in Japan was supported by the Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research—KAKENHI, grant numbers 15H05780 and 20H02989 to Y.K., and 20K15529 to S.K. Peer Reviewed "Article signat per 18 autors/es: Bastien Bissaro and Sayo Kodama and Takumi Nishiuchi and Anna Maria Díaz-Rovira and Hayat Hage and David Ribeaucourt and Mireille Haon and Sacha Grisel and A. Jalila Simaan and Fred Beisson and Stephanie M. Forget and Harry Brumer and Marie-Noëlle Rosso and Victor Guallar and Richard O’Connell and Mickaël Lafond and Yasuyuki Kubo and Jean-Guy Berrin "

This study aimed to clarify the success factor of snatch based on barbell trajectory and lifter's motion among elite male weightlifters. Motion analysis of snatch was conducted using digital videos recorded at the 2015 World Weightlifting Championships. Data on successful and unsuccessful snatch lifts of 22 lifters, each using the same weights, were analysed; the unsuccessful lift was due to a frontward barbell drop. Results revealed that the difference in backward barbell displacement between the turnover to catch phase (DxL) and peak backward barbell velocity (pVx-) was significantly greater in successful snatch than in frontward barbell drops (DxL: p 0.001, d 5.0, pVx-: p 0.01, d 2.0). Backward displacement of the lifters' centre of mass (COM) between the transition to turnover phase in a successful snatch lift was significantly smaller than that in an unsuccessful lift (p 0.05, d 2.0). It is considered that there was excessive backward leaning during unsuccessful lifts. However, no significant difference in maximum barbell height (Dy1) was found. Based on these findings, DxL and pVx- are success factors for snatch, whereas Dy1 is not. It is suggested that avoiding excessive backward-leaning of the body in the turnover phase may vary the chances of successful snatch among elite male weightlifters.

The objective of this study was to develop a comprehensive method for musculoskeletal simulation in swimming. In the developed method, the motion capture data of a swimming motion were first transformed into input data for the swimming human simulation model Swimming Human Model (SWUM). Then, the output data from SWUM were transformed into input data for the musculoskeletal simulation. In order to examine the validity of the developed method, the musculoskeletal simulations of the crawl and breaststroke by two swimmers were conducted. Based on the results of the crawl, the muscles of the upper limbs were activated significantly at the pull and push phases. The swimmer also adjusted the swimming speed, not by changing the effort of the upper limbs during the pull and push phases but by changing the length of the entry phase and the effort of the flutter kick. The results of the breaststroke showed that the muscles of the lower limbs were activated significantly at the kick phase. The swimmer adjusted the swimming speed, not by changing the effort of the upper limbs but by changing the length of the glide phase and the effort of kicking. The results from this study were consistent with the findings in previous studies, except for the period of large (nearly 180 degrees) flexion or abduction at the shoulder joint. The results also revealed the strategies of the swimmers to adjust their swimming speed. This indicates the promising potential of the musculoskeletal simulation, that is, it will be possible to estimate the strategy of a swimmer by conducting the simulation.

To breach the plant cuticle, many plant pathogenic fungi differentiate specialized infection structures (appressoria). In Colletotrichum orbiculare (cucumber anthracnose fungus), this differentiation requires unique proper G1 /S phase progression, regulated by two-component GTPase activating protein CoBub2/CoBfa1 and GTPase CoTem1. Since their homologues regulate mitotic exit, cytokinesis, or septum formation from yeasts to mammals, we asked whether the BUB2 function in G1 /S progression is specific to plant pathogenic fungi. Colletotrichum higginsianum and Magnaporthe oryzae were genetically analyzed to investigate conservation of BUB2 roles in cell cycle regulation, septum formation, and virulence. Expression profile of cobub2Δ was analyzed using a custom microarray. In bub2 mutants of both fungi, S phase initiation was earlier, and septum formation coordinated with a septation initiation network protein and contractile actin ring was impaired. Earlier G1 /S transition in cobub2Δ results in especially high expression of DNA replication genes and differing regulation of virulence-associated genes that encode proteins such as carbohydrate-active enzymes and small secreted proteins. The virulence of chbub2Δ and mobub2Δ was significantly reduced. Our evidence shows that BUB2 regulation of G1 /S transition and septum formation supports its specific requirement for appressorium development in plant pathogenic fungi.

Copper radical oxidases (CRO) form a class of enzymes with a longstanding history encompassing diverse substrate specificities. While the biological function of most CROs remains unknown, we observed that CROs active on aliphatic alcohols are found only in fungal plant pathogens. Here, we unveil the role of these CROs and the identity of their natural redox partner, a haem-iron peroxidase. Combining multiscale approaches, we report that Colletotrichum and Magnaporthe appressoria (specialized cells that puncture the plant cuticles) co-secrete this pair of metalloenzymes early during penetration. We show in vivo that mutant appressoria lacking either or both enzymes have impaired penetration ability and pathogenicity. We reveal in vitro a finely-tuned enzyme interplay is responsible for the oxidation of plant cuticular long-chain alcohols into aldehyde products, suggested to act as key molecular signals in the fungal infection machinery. Our results open new avenues to design oxidase-specific inhibitors as anti-penetrants for crop protection.

Center of mass (CoM) velocity variation in swimming direction is related to swimming performance and efficiency. However, it is difficult to calculate the CoM velocity during swimming. Therefore, we aimed to establish a practical estimation method for the CoM velocity in swimming direction during front crawl swimming with underwater cameras. Ten swimmers were recorded during front crawl swimming (25 m, maximal effort) using a motion capture system with 18 underwater and 9 land cameras. Three CoM velocity estimation methods were constructed (single-hip velocity, both-hips velocity, and both-hips velocity with simulated arm velocity correction). Each model was validated against the actual CoM velocity. The difference between the single-hip velocity and the actual CoM velocity in swimming direction was significantly larger compared with that of the other 2 models. Furthermore, the accuracy of CoM velocity estimation was increased when both-hips velocity was corrected using the simulated arm velocity. The method allowed estimation of the CoM velocity with only 2 underwater cameras with a maximal difference of 0.06 m·s−1. This study established a novel and practical method for the estimation of the CoM velocity in swimming direction during front crawl swimming.

Colletotrichum orbiculare species complex fungi are hemibiotrophic plant pathogens that cause anthracnose of field crops and weeds. Members of this group have genomes that are remarkably expanded relative to other Colletotrichum fungi and compartmentalized into AT-rich, gene-poor and GC-rich, gene-rich regions. Here, we present an updated version of the C. orbiculare genome, as well as draft genomes of three other members from the C. orbiculare species complex: the alfalfa pathogen C. trifolii, the prickly mallow pathogen C. sidae, and the burweed pathogen C. spinosum. The data reported here will be important for comparative genomics analyses to identify factors that play a role in the evolution and maintenance of the expanded, compartmentalized genomes of these fungi, which may contribute to their pathogenicity.

Currently, a strong public and political demand for reducing chemical pesticides exists, supporting strengthening of biological plant protection programs in agriculture. Biological plant protection strategies largely depend on introducing antagonistic microorganisms to agro-ecosystems. This approach would indeed reduce the impact of synthetic chemistries and may help avoiding development of fungicide-resistant pathogen populations, but on the other hand may strongly increase the load of microbial toxins. In crops treated with biological control agents, neither the newly forming (confronting) microbial communities nor the secondary metabolites synthesized by these are known. Many microbial secondary metabolites may not only exhibit acute toxicities significantly exceeding those of fungicides, but even act as strong carcinogens. This paper discusses the risks imposed by the introduction of biological control agents and strongly suggests including secondary metabolite gene cluster expression data into the list of information required for approving any plant protection agents and releasing them to the field. Secondary metabolism gene expression data could strongly support assessing the risk imposed by microorganisms used in disease and pest control.

The success factor of the snatch has not been identified. Determining the success factors of the snatch among elite weightlifters might help to attain a successful snatch. This study aimed at clarifying the factors that lead to a successful snatch based on barbell trajectory among elite male weightlifters. Data were collected at the 2017 World and Junior World Weightlifting Championships. We digitized the barbell trajectory of the successful and unsuccessful snatch attempts of 61 lifters&mdash, an unsuccessful lift would be as a result of a frontward barbell drop&mdash, and calculated the kinematic and kinetic parameters of the barbell. No significant difference was found in the barbell maximum height (Dy1) between the successful and unsuccessful lifts. The amount of backward displacement of the barbell in the second pull phase to the catch position (DxL) of the successful lift was significantly larger than that of the unsuccessful lift (successful: 0.11 &plusmn, 0.05 m, unsuccessful: 0.10 &plusmn, 0.06 m, p &lt, 0.01, d = 0.278). The barbell drop distance in the catch phase (Dy3) of the successful lift was significantly smaller than that in the unsuccessful lift (successful: 0.17 &plusmn, 0.04 m, unsuccessful: 0.18 &plusmn, 0.001, d = 0.361). These results suggest that DxL and Dy3 are factors leading to a successful snatch lift, but not Dy1. The relative position in the sagittal axis of the barbell and the lifter in the catch position, and catching the barbell when its momentum was low, are important in order to achieve a successful snatch.

Bacterial soft rot caused by Pectobacterium carotovorum subsp. carotovorum is one of the most important diseases of Chinese cabbage. To develop a microbial pesticide that is not only safe but also easily acceptable by consumers, we screened strains of lactic acid bacteria for biocontrol potential. One of 1166 isolates, Lactobacillus plantarum strain BY significantly reduced soft rot disease severity in Chinese cabbage in six different fields in Japan. This isolate also suppressed disease in onion, potato, and tomato. BY populations persisted on wounded Chinese cabbage leaves after spraying. Moreover, BY inhibits pathogen growth in an antibiosis assay. These results strongly suggest that BY inhibits the invasion of the pathogen at wounded sites on leaves and its proliferation in host tissues. This is the first report indicating the application of lactic acid bacteria for biological control of a plant disease under field conditions.

The hemibiotrophic fungus Colletotrichum orbiculare forms appressoria as infection structures and primarily establishes biotrophic infection in cucumber epidermal cells. Subsequently, it develops necrotrophic infection. In the pre-invasion stage, morphogenesis of appressoria of C. orbiculare is triggered by signals from the plant surface. We found that C. orbiculare PAG1 (Perish-in-the-Absence-of- GYP1 ), a component of MOR [morphogenesis-related NDR (nuclear Dbf2-related) kinase network] plays an essential role as a key component of the plant-specific signaling pathway and that hydrolysis of cutin by a spore surface esterase creates a cutin monomer that constitutes a key plant-derived signal. Development of the infection structure of C. orbiculare is strictly regulated by the cell cycle and we found that proper regulation of G1/S progression via two-component GAP genes, consisting of BUB2 (Budding-Uninhibited-by-Benomyl-2) and BFA1 (Byr-Four-Alike-1) is essential for the establishment of successful infection. In the post-invasion stage, the establishment of the biotrophic phase of hemibiotrophic fungi is crucial for successful infection. We found that C. orbiculare WHI2 (WHIsky-2), an Saccharomyces cerevisiae stress regulator homolog, is involved in the phase transition from biotrophy to necrotrophy through TOR (Target of Rapamycin) signaling, and is thus essential for full pathogenesis.

The cucumber anthracnose fungus Colletotrichum orbiculare forms a specialized infection structure, called an appressorium. Appressorium differentiation relies on fungal perception of physical and biochemical signals at the plant surface. Our previous report showed that the morphogenesis-related NDR (nuclear Dbf2-related) kinase pathway (MOR) is crucial for translating plant-derived signals for appressorium development. Here, we focused on identifying transcriptional regulators downstream of MOR that are involved in plant signal sensing and transduction for appressorium development. Based on whole-genome transcript profiling, we identified a Zn(II)2Cys6 transcription factor, CoMTF4, as a potential downstream factor of MOR. CoMTF4 was expressed in planta rather than in vitro under the control of the NDR kinase CoCbk1. Phenotypes of comtf4 mutants, strains with constitutively active CoCbk1 and strains with constitutive overexpression of CoMTF4 suggested that CoMtf4 acts downstream of MOR. Furthermore, nuclear localization of CoMtf4 was dependent on the MOR and responsive to plant-derived signals that lead to appressorium morphogenesis. Thus, we conclude that CoMtf4 is a transcription factor downstream of MOR that is essential for appressorium morphogenesis and pathogenesis and is regulated in response to plant-derived signals. This study provides insights into fungal sensing of plant signals and subsequent responses critical for appressorium formation.

To breach the plant cuticle, many plant pathogenic fungi differentiate specialized infection structures (appressoria). In Colletotrichum orbiculare (cucumber anthracnose fungus), this differentiation requires unique proper G

In contrast to the recent accomplishments of genome projects, cytological information on chromosomes and genomes of the genus Colletotrichum is very scarce. In this study, we performed mitotic cytological karyotyping for the three species, C. orbiculare, C. graminicola, and C. higginsianum by fluorescence microscopy and compared the results with those from genome projects. Chromosome number (CN) of C. orbiculare was determined for the first time to be n=10 with no minichromosomes (MCs) in the genome, while CNs of C. graminicola and C. higginsianum were consistent with those from their genome project including the number of MCs. Regarding chromosome features, C. orbiculare was peculiar in that each chromosome was distinctly partitioned into a highly AT-rich pericentromeric region and the remaining highly GC-rich regions, and the pericentromeric region was judged to be constitutive heterochromatin. Integrating all the discernible morphological characteristics such as chromosome length, nucleolar organizing region, and DAPI-stained regions, idiograms were constructed for the three species. The overall cytological features of the chromosomes and genomes fit well with the data from the genome projects in terms of genome size, GC-content, and the occurrence of AT-rich regions. This study represents the most comprehensive and detailed mitotic cytological karyotyping of fungi ever reported.

Plant infection by pathogenic fungi involves the differentiation of appressoria, specialized infection structures, initiated by fungal sensing and responding to plant surface signals. How plant fungal pathogens control infection-related morphogenesis in response to plant-derived signals has been unclear. Here we showed that the morphogenesis-related NDR kinase pathway (MOR) of the cucumber anthracnose fungus Colletotrichum orbiculare is crucial for appressorium development following perception of plant-derived signals. By screening of random insertional mutants, we identified that the MOR element CoPag1 (Perish-in-the-absence-of-GYP1) is a key component of the plant-derived signaling pathway involved in appressorium morphogenesis. Constitutive activation of the NDR kinase CoCbk1 (Cell-wall-biosynthesis-kinase-1) complemented copag1 defects. Furthermore, copag1 deletion impaired CoCbk1 phosphorylation, suggesting that CoPag1 functions via CoCbk1 activation. Searching for the plant signals that contribute to appressorium induction via MOR, we found that the cutin monomer n-octadecanal, degraded from the host cuticle by conidial esterases, functions as a signal molecule for appressorium development. Genome-wide transcriptional profiling during appressorium development revealed that MOR is responsible for the expression of a subset of the plant-signal-induced genes with potential roles in pathogenicity. Thus, MOR of C. orbiculare has crucial roles in regulating appressorium development and pathogenesis by communicating with plant-derived signals., Author summary Phytopathogenic fungi cause many of the most serious crop diseases. Many fungal pathogens form specialized infection structures, called appressoria in response to plant surface signals. Networks of protein kinase-based signaling pathways regulate a wide variety of key morphological processes including appressorium development in several fungal pathogens. However, the precise link between plant signals and fungal intracellular transduction is poorly understood. Here, we report on the identification of a native molecule and a cognate signal transduction pathway involved in appressorium morphogenesis of the cucumber anthracnose fungus Colletotrichum orbiculare. We demonstrate that the widely conserved morphogenesis-related NDR kinase pathway (MOR) of C. orbiculare regulates infection structure development triggered by plant-derived signals and involves in pathogenesis. The cutin monomer n-octadecanal released from the host cuticle by conidial esterases functions as the signal molecule for appressorium development via MOR. Inactivating MOR resulted in downregulation of the plant-signal-induced genes including fungal secreted proteins that potentially facilitate infection. Thus, MOR is the crucial coordinator connecting plant surface signals with infection-related morphogenesis and pathogenesis. While previous reports have revealed that MOR is crucial for controlling cell polarity and differentiation in other fungi, our study provides its new role in the interaction of fungal pathogens with host plant.

Colletotrichum orbiculare (syn. C. lagenarium) is the causal agent of anthracnose disease on cucurbit plants. This fungus forms dome-shaped, melanized appressoria as a host invasion structure. Strain 104-T (MAFF240422) of C. orbiculare, which was originally isolated from a cucumber plant in 1951 by Dr. Yasumori, Kyoto University, has proven to be an excellent experimental model for the study of fungal pathogenesis and morphogenesis because of its stable pathogenicity and synchronous infection-related morphogenesis. This review considers the discoveries made during 60 years of study on C. orbiculare. In particular, we focus on advances made in the last two decades, which have provided a basis for the molecular analysis not only of fungal morphogenesis, but also of plant–microbe interactions, including plant immunity to adapted and nonadapted Colletotrichum fungi. This substantial body of innovative research was originated by the Phytopathological Society of Japan and represents a major contribution to the international research communities working on plant pathology, plant–microbe interactions, and fungal molecular genetics. This review deals with the past achievements and future prospects in the study of Colletotrichum biology, focusing on the molecular genetics of C. orbiculare with regard to four aspects: (1) metabolic and functional development of infection structures, (2) signaling pathways required for fungal pathogenesis, (3) pathogen-associated molecular patterns (PAMPs) and host basal resistance, and (4) establishment of host specificity.

The hemibiotrophic fungus Colletotrichum orbiculare first establishes a biotrophic infection stage in cucumber (Cucumber sativus) epidermal cells and subsequently transitions to a necrotrophic stage. Here, we found that C. orbiculare established hemibiotrophic infection via C. orbiculare WHI2, a yeast stress regulator homolog, and TOR (target of rapamycin) signaling. Plant defense responses such as callose deposition, H2O2, and antimicrobial proteins were strongly induced by the C. orbiculare whi2Δ mutant, resulting in defective pathogenesis. Expression analysis of biotrophy-specific genes evaluated by the promoter VENUS fusion gene indicated weaker VENUS signal intensity in the whi2Δ mutant, thereby suggesting that C. orbiculare WHI2 plays a key role in regulating biotrophic infection of C. orbiculare. The involvement of CoWHI2 in biotrophic infection was further explored with a DNA microarray. In the Cowhi2Δ mutant, TOR-dependent ribosomal protein–related genes were strikingly upregulated compared with the wild type. Moreover, callose deposition in the host plant after inoculation with the Cowhi2Δ mutant treated with rapamycin, which inhibits TOR activity, was reduced, and the mutant remained biotrophic in contrast to the untreated mutant. Thus, regulation of TOR by Whi2 is apparently crucial to the biotrophic stage of hemibiotrophic infection in C. orbiculare.

The conidia of Colletotrichum orbiculare, the causal agent of cucumber anthracnose, develop appressoria that are pigmented with melanin for host plant infection. Premature appressoria contain abundant lipid droplets (LDs), but these disappear during appressorial maturation, indicating lipolysis inside the appressorial cells. The lipolysis and melanization in appressoria require the peroxin PEX6, suggesting the importance of peroxisomal metabolism in these processes. To investigate the relationships between appressorial lipolysis and fungal metabolic pathways, C. orbiculare knockout mutants of MFE1, which encodes a peroxisomal multifunctional enzyme, were generated in this study, and the phenotype of the mfe1 mutants was investigated. In contrast to the wild-type strain, which forms melanized appressoria, the mfe1 mutants formed colorless nonmelanized appressoria with abundant LDs, similar to those of pex6 mutants. This indicates that fatty acid β-oxidation in peroxisomes is critical for the appressorial melanization and lipolysis of C. orbiculare. Soraphen A, a specific inhibitor of acetyl-CoA carboxylase, inhibited appressorial lipolysis and melanization, producing phenocopies of the mfe1 mutants. This suggests that the conversion of acetyl-CoA, derived from fatty acid β-oxidation, to malonyl-CoA is required for the activation of lipolysis in appressoria. Surprisingly, we found that genetically blocking PKS1-dependent polyketide synthesis, an initial step in melanin biosynthesis, also impaired appressorial lipolysis. In contrast, genetically or pharmacologically blocking the steps in melanin synthesis downstream from PKS1 did not abolish appressorial lipolysis. These findings indicate that melanin biosynthesis, as well as fatty acid β-oxidation, is involved in the regulation of lipolysis inside fungal infection structures.

The hemibiotrophic ascomycete Colletotrichum higginsianum is the casual agent of anthracnose disease of cruciferous plants. High efficiency transformation by Agrobacterium tumefaciens-mediated gene transfer has been established for this fungus. However, targeted gene mutagenesis through homologous recombination rarely occurs in C. higginsianum. We have identified and disrupted the C. higginsianum homologue of the human Ku70 gene, ChKU70, which encodes a protein that plays a role in non-homologous end-joining for repair of DNA breaks. chku70 mutants showed a dramatic increase in the frequency of integration of introduced exogenous DNA fragments by homologous recombination without any detectable phenotypic defects. This result demonstrates that the chku70 mutant is an efficient recipient for targeted gene mutagenesis in C. higginsianum. We have also developed a novel approach [named direct repeat recombination-mediated gene targeting (DRGT)] for targeted gene disruption through Agrobacterium tumefaciens-mediated gene transfer. DRGT utilizes homologous recombination between repeated sequences on the T-DNA flanking a partial fragment of the target gene. Our results suggest that DRGT in the chku70 mutant background could be a useful tool for rapid isolation of targeted gene disruptants in C. higginsianum.

Peroxisomes are ubiquitous organelles of eukaryotic cells that fulfill a variety of biochemical functions, including β-oxidation of fatty acids. Here, we report that an ortholog of the Saccharomyces cerevisiae peroxisome biogenesis gene PEX13 is required for pathogenicity of Colletotrichum orbiculare. CoPEX13 was identified by screening random insertional mutants for deficiency in fatty acid utilization. Targeted knockout mutants of CoPEX13 were unable to utilize fatty acids as a carbon source. Expression analysis using green fluorescent protein fused to the peroxisomal targeting signals PTS1 and PTS2 revealed that the import machinery for peroxisomal matrix proteins was impaired in copex13 mutants. Appressoria formed by the copex13 mutants were defective in both melanization and penetration ability on host plants, had thin cell walls, and lacked peroxisomes. Moreover, the concentration of intracellular glycerol was lower in copex13 appressoria than those of the wild type. These findings indicate that fatty acid oxidation in peroxisomes is required not only for appressorium melanization but also for cell wall biogenesis and metabolic processes involved in turgor generation, all of which are essential for appressorium penetration ability.

Colletotrichum higginsianum is a fungal pathogen that infects a wide variety of cruciferous plants, causing important crop losses. We have used map-based cloning and natural variation analysis of 19 Arabidopsis ecotypes to identify a dominant resistance locus against C. higginsianum. This locus named RCH2 (for recognition of C. higginsianum) maps in an extensive cluster of disease-resistance loci known as MRC-J in the Arabidopsis ecotype Ws-0. By analyzing natural variations within the MRC-J region, we found that alleles of RRS1 (resistance to Ralstonia solanacearum 1) from susceptible ecotypes contain single nucleotide polymorphisms that may affect the encoded protein. Consistent with this finding, two susceptible mutants, rrs1-1 and rrs1-2, were identified by screening a T-DNA-tagged mutant library for the loss of resistance to C. higginsianum. The screening identified an additional susceptible mutant (rps4-21) that has a 5-bp deletion in the neighboring gene, RPS4-Ws, which is a well-characterized R gene that provides resistance to Pseudomonas syringae pv. tomato strain DC3000 expressing avrRps4 (Pst-avrRps4). The rps4-21/rrs1-1 double mutant exhibited similar levels of susceptibility to C. higginsianum as the single mutants. We also found that both RRS1 and RPS4 are required for resistance to R. solanacearum and Pst-avrRps4. Thus, RPS4-Ws and RRS1-Ws function as a dual resistance gene system that prevents infection by three distinct pathogens.

Plant basal resistance is activated by virulent pathogens in susceptible host plants. A Colletotrichum orbiculare fungal mutant defective in the SSD1 gene, which regulates cell wall composition, is restricted by host basal resistance responses. Here, we identified the Nicotiana benthamiana signaling pathway involved in basal resistance by silencing the defense-related genes required for restricting the growth of the C. orbiculare mutant. Only silencing of MAP Kinase Kinase2 or of both Salicylic Acid Induced Protein Kinase (SIPK) and Wound Induced Protein Kinase (WIPK), two mitogen-activated protein (MAP) kinases, allowed the mutant to infect and produce necrotic lesions similar to those of the wild type on inoculated leaves. The fungal mutant penetrated host cells to produce infection hyphae at a higher frequency in SIPK WIPK-silenced plants than in nonsilenced plants, without inducing host cellular defense responses. Immunocomplex kinase assays revealed that SIPK and WIPK were more active in leaves inoculated with mutant fungus than with the wild type, suggesting that induced resistance correlates with MAP kinase activity. Infiltration of heat-inactivated mutant conidia induced both SIPK and WIPK more strongly than did those of the wild type, while conidial exudates of the wild type did not suppress MAP kinase induction by mutant conidia. Therefore, activation of a specific MAP kinase pathway by fungal cell surface components determines the effective level of basal plant resistance.

Kelch repeat proteins are important mediators of fundamental cellular functions and are found in diverse organisms. However, the roles of these proteins in filamentous fungi have not been characterized. We isolated a kelch repeat-encoding gene of Colletotrichum lagenarium ClaKEL2 , a Schizosaccharomyces pombe tea1 homologue. Analysis of the clakel2 mutant indicated that ClaKEL2 was required for the establishment of cellular polarity essential for proper morphogenesis of appressoria and that there is a plant signal-specific bypass pathway for appressorium development which circumvents ClaKEL2 function. Clakel2p was localized in the polarized region of growing hyphae and germ tubes, and the localization was disturbed by a microtubule assembly blocker. The clakel2 mutants formed abnormal appressoria, and those appressoria were defective in penetration hypha development into cellulose membranes, an artificial model substrate for fungal infection. Surprisingly, the clakel2 mutants formed normal appressoria on the host plant and retained penetration ability. Normal appressorium formation on the artificial substrate by the clakel2 mutants was restored when cells were incubated in the presence of CaCl 2 or exudates from cucumber cotyledon. Furthermore, calcium channel modulators inhibited restoration of normal appressorium formation. These results suggest that there could be a bypass pathway that transduces a plant-derived signal for appressorium development independent of ClaKEL2 and that a calcium signal is involved in this transduction pathway.

Summary Fungal plant pathogens have evolved diverse strate- gies to overcome the multilayered plant defence responses that confront them upon host invasion. Here we show that pathogenicity of the cucumber anthracnose fungus, Colletotrichum lagenarium, and the rice blast fungus, Magnaporthe grisea, requires a gene orthologous to Saccharomyces cerevisiae SSD1, a regulator of cell wall assembly. Screening for C. lagenarium insertional mutants deficient in patho- genicity led to the identification of ClaSSD1. Follow- ing targeted gene replacement, appressoria of classd1 mutants retained the potential for penetration but were unable to penetrate into host epidermal cells. Transmission electron microscopy suggested that appressorial penetration by classd1 mutants was restricted by plant cell wall-associated defence responses, which were observed less frequently with the wild-type strain. Interestingly, on non-host onion epidermis classd1 mutants induced papilla formation faster and more abundantly than the wild type. Simi- larly, colonization of rice leaves by M. grisea was severely reduced after deletion of the orthologous MgSSD1 gene and attempted infection by the mutants was accompanied by the accumulation of reactive oxygen species within the host cell. These results suggest that appropriate assembly of the fungal cell wall as regulated by SSD1 allows these pathogens to establish infection by avoiding the induction of host defence responses.

Morphogenesis in filamentous fungi depends on appropriate cell cycle progression. Here, we report that cells of the cucumber anthracnose fungus Colletotrichum orbiculare regulate G1/S progression via a two-component GAP, consisting of Budding-uninhibited-by-benomyl-2 (Bub2) and Byr-four-alike-1 (Bfa1) as well as its GTPase Termination-of-M-phase-1 (Tem1) to establish successful infection. In a random insertional mutagenesis screen of infection-related morphogenesis, we isolated a homolog of Saccharomyces cerevisiae, BUB2, which encodes a two-component Rab GAP protein that forms a GAP complex with Bfa1p and negatively regulates mitotic exit. Interestingly, disruption of either Co BUB2 or Co BFA1 resulted in earlier onset of nuclear division and decreased the time of phase progression from G1 to S during appressorium development. S. cerevisiae GTPase Tem1p is the downstream target of the Bub2p/Bfa1p GAP complex. Introducing the dominant-negative form of Co Tem1 into Co bub2Δ or Co bfa1Δ complemented the defect in G1/S progression, indicating that Co Bub2/Co Bfa1 regulates G1/S progression via Co Tem1. Based on a pathogenicity assay, we found that Co bub2Δ and Co bfa1Δ reduced pathogenesis by attenuating infection-related morphogenesis and enhancing the plant defense response. Thus, during appressorium development, C. orbiculare Bub2/Bfa1 regulates G1/S progression via Co Tem1, and this regulation is essential to establish plant infection.

The cucumber anthracnose fungus Colletotrichum orbiculare forms specialized cells called appressoria for host penetration. We identified a gene, FAM1, encoding a novel peroxin protein that is essential for peroxisome biogenesis and that associates with Woronin bodies (WBs), dense-core vesicles found only in filamentous ascomycete fungi which function to maintain cellular integrity. The fam1 disrupted mutants were unable to grow on medium containing oleic acids as the sole carbon source and were nonpathogenic, being defective in both appressorium melanization and host penetration. Fluorescent proteins carrying peroxisomal targeting signals (PTSs) were not imported into the peroxisomes of fam1 mutants, suggesting that FAM1 is a novel peroxisomal biogenesis gene (peroxin). FAM1 did not show significant homology to any Saccharomyces cerevisiae peroxins but resembled conserved filamentous ascomycete-specific Pex22-like proteins which contain a predicted Pex4-binding site and are potentially involved in recycling PTS receptors from peroxisomes to the cytosol. C. orbiculare FAM1 complemented the peroxisomal matrix protein import defect of the S. cerevisiae pex22 mutant. Confocal microscopy of Fam1-GFP (green fluorescent protein) fusion proteins and immunoelectron microscopy with anti-Fam1 antibodies showed that Fam1 localized to nascent WBs budding from peroxisomes and mature WBs. Association of Fam1 with WBs was confirmed by colocalization with WB matrix protein CoHex1 (C. orbiculare Hex1) and WB membrane protein CoWsc (C. orbiculare Wsc) and by subcellular fractionation and Western blotting with antibodies to Fam1 and CoHex1. In WB-deficient cohex1 mutants, Fam1 was redirected to the peroxisome membrane. Our results show that Fam1 is a WB-associated peroxin required for pathogenesis and raise the possibility that localized receptor recycling occurs in WBs., IMPORTANCE Colletotrichum orbiculare is a fungus causing damaging disease on Cucurbitaceae plants. In this paper, we characterize a novel peroxisome biogenesis gene from this pathogen called FAM1. Although no genes with significant homology are present in Saccharomyces cerevisiae, FAM1 contains a predicted Pex4-binding site typical of Pex22 proteins, which function in the recycling of PTS receptors from peroxisomes to the cytosol. We show that FAM1 complements the defect in peroxisomal matrix protein import of S. cerevisiae pex22 mutants and that fam1 mutants are completely defective in peroxisome function, fatty acid metabolism, and pathogenicity. Remarkably, we found that this novel peroxin is specifically localized on the bounding membrane of Woronin bodies, which are small peroxisome-derived organelles unique to filamentous ascomycete fungi that function in septal pore plugging. Our finding suggests that these fungi have coopted the Woronin body for localized receptor recycling during matrix protein import.