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.

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.

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.

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.

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.

A Biomechanical Comparison of Successful and Unsuccessful Snatch Attempts among Elite Male Weightlifters.

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-an unsuccessful lift would be as a result of a frontward barbell drop-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 ± 0.05 m; unsuccessful: 0.10 ± 0.06 m; p < 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 ± 0.04 m; unsuccessful: 0.18 ± 0.04 m; p < 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.

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 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.

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.

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.

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.

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.

Colletotrichum orbiculare FAM1 Encodes a Novel Woronin Body-Associated Pex22 Peroxin Required for Appressorium-Mediated Plant Infection.

UNLABELLED: 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.

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.

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.

Ras GTPase activating protein CoIra1 is involved in infection-related morphogenesis by regulating cAMP and MAPK signaling pathways through CoRas2 in Colletotrichum orbiculare.

Colletotrichum orbiculare is the causative agent of anthracnose disease on cucurbitaceous plants. Several signaling pathways, including cAMP-PKA and mitogen-activating protein kinase (MAPK) pathways are involved in the infection-related morphogenesis and pathogenicity of C. orbiculare. However, upstream regulators of these pathways for this species remain unidentified. In this study, CoIRA1, encoding RAS GTPase activating protein, was identified by screening the Agrobacterium tumefaciens-mediated transformation (AtMT) mutant, which was defective in the pathogenesis of C. orbiculare. The coira1 disrupted mutant showed an abnormal infection-related morphogenesis and attenuated pathogenesis. In Saccharomyces cerevisiae, Ira1/2 inactivates Ras1/2, which activates adenylate cyclase, leading to the synthesis of cAMP. Increase in the intracellular cAMP levels in coira1 mutants and dominant active forms of CoRAS2 introduced transformants indicated that CoIra1 regulates intracellular cAMP levels through CoRas2. Moreover, the phenotypic analysis of transformants that express dominant active form CoRAS2 in the comekk1 mutant or a dominant active form CoMEKK1 in the coras2 mutant indicated that CoRas2 regulates the MAPK CoMekk1-Cmk1 signaling pathway. The CoRas2 localization pattern in vegetative hyphae of the coira1 mutant was similar to that of the wild-type, expressing a dominant active form of RFP-CoRAS2. Moreover, we demonstrated that bimolecular fluorescence complementation (BiFC) signals between CoIra1 and CoRas2 were detected in the plasma membrane of vegetative hyphae. Therefore, it is likely that CoIra1 negatively regulates CoRas2 in vegetative hyphae. Furthermore, cytological analysis of the localization of CoIraI and CoRas2 revealed the dynamic cellular localization of the proteins that leads to proper assembly of F-actin at appressorial pore required for successful penetration peg formation through the pore. Thus, our results indicated that CoIra1 is involved in infection-related morphogenesis and pathogenicity by proper regulation of cAMP and MAPK signaling pathways through CoRas2.

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).

Breaking restricted taxonomic functionality by dual resistance genes.

NB-LRR-type disease resistance (R) genes have been used in traditional breeding programs for crop protection. However, functional transfer of NB-LRR-type R genes to plants in taxonomically distinct families to establish pathogen resistance has not been successful. Here we demonstrate that a pair of Arabidopsis (Brassicaceae) NB-LRR-type R genes, RPS4 and RRS1, properly function in two other Brassicaceae, Brassica rapa and B. napus, but also in two Solanaceae, Nicotiana benthamiana and tomato (Solanum lycopersicum). The solanaceous plants transformed with RPS4/RRS1 confer bacterial effector-specific immunity responses. Furthermore, RPS4 and RRS1, which confer resistance to a fungal pathogen Colletotrichum higginsianum in Brassicaceae, also protect against Colletotrichum orbiculare in cucumber (Cucurbitaceae). Thus the successful transfer of two R genes at the family level overcomes restricted taxonomic functionality. This implies that the downstream components of R genes must be highly conserved and interfamily utilization of R genes can be a powerful strategy to combat pathogens.

Interfamily transfer of dual NB-LRR genes confers resistance to multiple pathogens.

A major class of disease resistance (R) genes which encode nucleotide binding and leucine rich repeat (NB-LRR) proteins have been used in traditional breeding programs for crop protection. However, it has been difficult to functionally transfer NB-LRR-type R genes in taxonomically distinct families. Here we demonstrate that a pair of Arabidopsis (Brassicaceae) NB-LRR-type R genes, RPS4 and RRS1, properly function in two other Brassicaceae, Brassica rapa and Brassica napus, but also in two Solanaceae, Nicotiana benthamiana and tomato (Solanum lycopersicum). The solanaceous plants transformed with RPS4/RRS1 confer bacterial effector-specific immunity responses. Furthermore, RPS4 and RRS1, which confer resistance to a fungal pathogen Colletotrichum higginsianum in Brassicaceae, also protect against Colletotrichum orbiculare in cucumber (Cucurbitaceae). Importantly, RPS4/RRS1 transgenic plants show no autoimmune phenotypes, indicating that the NB-LRR proteins are tightly regulated. The successful transfer of two R genes at the family level implies that the downstream components of R genes are highly conserved. The functional interfamily transfer of R genes can be a powerful strategy for providing resistance to a broad range of pathogens.