The disordered C-terminal tail of fungal LPMOs from phytopathogens mediates protein dimerization and impacts plant penetration.

Lytic polysaccharide monooxygenases (LPMOs) are monocopper enzymes that oxidatively degrade various polysaccharides, such as cellulose. Despite extensive research on this class of enzymes, the role played by their C-terminal regions predicted to be intrinsically disordered (dCTR) has been overlooked. Here, we investigated the function of the dCTR of an LPMO, called CoAA9A, up-regulated during plant infection by Colletotrichum orbiculare, the causative agent of anthracnose. After recombinant production of the full-length protein, we found that the dCTR mediates CoAA9A dimerization in vitro, via a disulfide bridge, a hitherto-never-reported property that positively affects both binding and activity on cellulose. Using SAXS experiments, we show that the homodimer is in an extended conformation. In vivo, we demonstrate that gene deletion impairs formation of the infection-specialized cell called appressorium and delays penetration of the plant. Using immunochemistry, we show that the protein is a dimer not only in vitro but also in vivo when secreted by the appressorium. As these peculiar LPMOs are also found in other plant pathogens, our findings open up broad avenues for crop protection.

Colletotrichum orbiculare strains distributed in Japan: race identification and evaluation of virulence to cucurbits.

The pathogen Colletotrichum orbiculare is causal fungus of cucurbit anthracnose. Multiple races have been identified in the United States, suggesting that it is necessary to cultivate suitable resistant cultivars and breed new cultivars with the most suitable resistance gene. This study examined the pathogenicity and virulence of 20 strains in Japan to clarify the existence of races and virulence differences. Based on the symptoms on inoculated cotyledons and true leaves of watermelon, we could evaluate the compatibility of each strain to each host cultivar. Our analysis based on the reaction to the host cultivar harboring the resistance gene Ar-1 (Cla001017) revealed the existence of three races in Japan. An alarming result was that a race that overcame Ar-1, which is a target gene in current watermelon breeding in Japan, is present in Japan. The cucumber and melon host cultivars showed diverse symptoms, whereas a squash cultivar was resistant to all strains. Three strains caused severe damage even to the most resistant cucumber cultivar 'Ban Kyuri' and resistant cultivars harboring Cssgr, a well-known gene conferring loss-of-susceptibility resistance. Screening genetic resources for novel resistance genes using strains with high virulence is of vital importance for watermelon, cucumber, and melon production.

Colletotrichum orbiculare MTF4 Is a Key Transcription Factor Downstream of MOR Essential for Plant Signal-Dependent Appressorium Development and 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.

The morphogenesis-related NDR kinase pathway of Colletotrichum orbiculare is required for translating plant surface signals into infection-related morphogenesis and pathogenesis.

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.

Plant pathogenic fungi Colletotrichum and Magnaporthe share a common G1 phase monitoring strategy for proper appressorium development.

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.

Colletotrichum orbiculare Regulates Cell Cycle G1/S Progression via a Two-Component GAP and a GTPase to Establish Plant Infection.

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.

A Practical Estimation Method for Center of Mass Velocity in Swimming Direction During Front Crawl Swimming.

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 WHI2, a Yeast Stress-Response Regulator Homolog, Controls the Biotrophic Stage of Hemibiotrophic Infection Through TOR Signaling.

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.

Functional Role of the Front and Back Legs During a Track Start with Special Reference to an Inverted Pendulum Model in College Swimmers.

This study investigated factors that determine the velocity of the center of mass (CM) and flight distance from a track start to devise effective technical and physical training methods. Nine male and 5 female competitive swimmers participated in this study. Kinematics and ground reaction forces of the front and back legs were recorded using a video camera and force plates. The track start was modeled as an inverted pendulum system including a compliant leg, connecting the CM and front edge of the starting block. The increase in the horizontal velocity of the CM immediately after the start signal was closely correlated with the rotational component of the inverted pendulum. This rotational component at hands-off was significantly correlated with the average vertical force of the back plate from the start signal to hands-off (r = .967, P < .001). The flight distance / height was significantly correlated with the average vertical force of the front plate from the back foot-off to front foot-off (r = .783, P < .01). The results indicate that the legs on the starting block in the track start play a different role in the behavior of the inverted pendulum.

Cytological analyses of the karyotypes and chromosomes of three Colletotrichum species, C. orbiculare, C. graminicola and C. higginsianum.

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.

Vascular plant one-zinc-finger protein 1/2 transcription factors regulate abiotic and biotic stress responses in Arabidopsis.

Plants adapt to abiotic and biotic stresses by activating abscisic acid-mediated (ABA) abiotic stress-responsive and salicylic acid-(SA) or jasmonic acid-mediated (JA) biotic stress-responsive pathways, respectively. Although the abiotic stress-responsive pathway interacts antagonistically with the biotic stress-responsive pathways, the mechanisms that regulate these pathways remain largely unknown. In this study, we provide insight into the function of vascular plant one-zinc-finger proteins (VOZs) that modulate various stress responses in Arabidopsis. The expression of many stress-responsive genes was changed in the voz1voz2 double mutant under normal growth conditions. Consistent with altered stress-responsive gene expression, freezing- and drought-stress tolerances were increased in the voz1voz2 double mutant. In contrast, resistance to a fungal pathogen, Colletotrichum higginsianum, and to a bacterial pathogen, Pseudomonas syringae, was severely impaired. Thus, impairing VOZ function simultaneously conferred increased abiotic tolerance and biotic stress susceptibility. In a chilling stress condition, both the VOZ1 and VOZ2 mRNA expression levels and the VOZ2 protein level gradually decreased. VOZ2 degradation during cold exposure was completely inhibited by the addition of the 26S proteasome inhibitor, MG132, a finding that suggested that VOZ2 degradation is dependent on the ubiquitin/26S proteasome system. In voz1voz2, ABA-inducible transcription factor CBF4 expression was enhanced significantly even under normal growth conditions, despite an unchanged endogenous ABA content. A finding that suggested that VOZs negatively affect CBF4 expression in an ABA-independent manner. These results suggest that VOZs function as both negative and positive regulators of the abiotic and biotic stress-responsive pathways, and control Arabidopsis adaptation to various stress conditions.

Function of peroxisomes in plant-pathogen interactions.

Peroxisomes are ubiquitous organelles of eukaryotic cells that accomplish a variety of biochemical functions, including ß-oxidation of fatty acids, glyoxylate cycle, etc. Many reports have been accumulating that indicate peroxisome related metabolic functions are essential for pathogenic development of plant pathogenic fungi. They include peroxisome biogenesis proteins, peroxins and preferential destruction of peroxisomes, pexophagy. Gene disrupted mutants of anthracnose disease pathogen Colletotrichum orbiculare or rice blast pathogen Magnaporthe oryzae defective in peroxins or pexophagy showed deficiency in pathogenesis. Woronin body, a peroxisome related cellular organelle that is related to endurance of fungal cells against environmental damage has essential roles in pathogenesis of M. oryzae. Also, peroxisome related metabolisms such as ß-oxidation and glyoxylate cycle are essential for pathogenesis in several plant pathogenic fungi. In addition, secondary metabolisms including polyketide melanin biosynthesis of C. orbiculare and M. oryzae, and host selective toxins produced by necrotrophic pathogen Alternaria alternata have pivotal roles in fungal pathogenesis. Every such factor was listed and their functions for pathogenesis were demonstrated (Table 18.1 and Fig. 18.1).

Comparative genomic and transcriptomic analyses reveal the hemibiotrophic stage shift of Colletotrichum fungi.

Hemibiotrophic fungal plant pathogens represent a group of agronomically significant disease-causing agents that grow first on living tissue and then cause host death in later, necrotrophic growth. Among these, Colletotrichum spp. are devastating pathogens of many crops. Identifying expanded classes of genes in the genomes of phytopathogenic Colletotrichum, especially those associated with specific stages of hemibiotrophy, can provide insights on how these pathogens infect a large number of hosts. The genomes of Colletotrichum orbiculare, which infects cucurbits and Nicotiana benthamiana, and C. gloeosporioides, which infects a wide range of crops, were sequenced and analyzed, focusing on features with potential roles in pathogenicity. Regulation of C. orbiculare gene expression was investigated during infection of N. benthamiana using a custom microarray. Genes expanded in both genomes compared to other fungi included sequences encoding small, secreted proteins (SSPs), secondary metabolite synthesis genes, proteases and carbohydrate-degrading enzymes. Many SSP and secondary metabolite synthesis genes were upregulated during initial stages of host colonization, whereas the necrotrophic stage of growth is characterized by upregulation of sequences encoding degradative enzymes. Hemibiotrophy in C. orbiculare is characterized by distinct stage-specific gene expression profiles of expanded classes of potential pathogenicity genes.

Biomechanical comparison of successful snatch and unsuccessful frontward barbell drop in world-class male weightlifters.

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.

Primary and secondary metabolism regulates lipolysis in appressoria of Colletotrichum 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 ß-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 ß-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 ß-oxidation, is involved in the regulation of lipolysis inside fungal infection structures.

Niemann-Pick Type C Proteins Are Required for Sterol Transport and Appressorium-Mediated Plant Penetration of Colletotrichum orbiculare.

Many biotrophic and hemibiotrophic fungal pathogens use appressoria to directly penetrate the host plant surface. In the cucumber anthracnose fungus Colletotrichum orbiculare, differentiation of appressoria requires a proper G1/S cell cycle progression, regulated by the GTPase-activating protein complex CoBub2-CoBfa1 and its downstream GTPase CoTem1. To explore the mechanisms by which the CoTem1 cascade regulates plant infection, we screened for CoTem1 interaction factors and identified a Niemann-Pick type C2 homolog (CoNpc2). Niemann-Pick type C proteins NPC1 and NPC2 are sterol-binding proteins required for sterol export from lysosomes (vacuoles) in humans and yeasts. We showed that CoNpc2 colocalized with CoNpc1 in late endosomes and vacuoles and that disruption of its gene resulted in aberrant sterol accumulation in vacuoles and loss of sterol membrane localization, indicating that NPC proteins are engaged in sterol transport in C. orbiculare. For appressorium infection, sterol transport and proper distribution mediated by CoNpc1 and CoNpc2 are critical for membrane integrity and membrane curvature with actin assembly, leading to penetration peg emergence and appressorial cone formation. Our results revealed a novel mechanism by which NPC proteins regulate appressorium-mediated plant infection. IMPORTANCE Fungal morphogenesis requires accurate cell cycle progression. Two-component GTPase-activating protein (GAP) CoBub2-CoBfa1 interacts with downstream GTPase CoTem1 and is required for G1/S progression to establish plant infection in Colletotrichum orbiculare. To understand the pathogenicity related functions of CoTem1 downstream, we identified a Niemann-Pick type C2 homolog (CoNpc2) as a novel physical interaction factor with CoTem1. Whereas NPC proteins (NPC1 and NPC2) are essential for sterol homeostasis in humans and yeasts, their functions in plant invasion by pathogenic fungi have remained unclear. In this study, we show that CoNPC1 and CoNPC2 play a critical role in intracellular sterol transport and that appropriate sterol distribution is required for membrane integrity and membrane curvature with actin assembly that leads to appressorium-mediated plant penetration and pathogenicity of C. orbiculare. Our findings suggest the importance of sterol distribution in fungal morphogenesis during plant infection.

Tandem metalloenzymes gate plant cell entry by pathogenic fungi.

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.

RRS1 and RPS4 provide a dual Resistance-gene system against fungal and bacterial pathogens.

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.

A Kelch repeat protein, Cokel1p, associates with microtubules and is involved in appressorium development in Colletotrichum orbiculare.

Kelch repeat proteins are conserved in diverse organisms and some are known to mediate fundamental cellular functions. We isolated the gene CoKEL1, encoding a novel kelch repeat protein, from Colletotrichum orbiculare. Analysis of a cokel1 mutant indicated that CoKEL1 is involved in proper appressorium development and cell wall synthesis. Appressoria produced by cokel1 disruption mutants showed irregular shape and impairment of turgor generation and the mutant appressoria rarely penetrated to form infection hyphae in host epidermal cells. Accordingly, cokel1 mutants had reduced pathogenicity on host leaves compared with the wild type. Furthermore, the cokel1 mutant was more sensitive to cell-wall-degrading enzymes and showed altered labeling with the cell wall stain Calcofluor white. Cokel1p was localized on cortical and spindle microtubules in vegetative hyphae. These results suggest that Cokel1p is a microtubule-associated protein involved in infection-related morphogenesis and pathogenicity. This is the first report that a kelch repeat protein is required for the pathogenicity of a fungal plant pathogen.