Search

The hemibiotrophic fungal plant pathogen Colletotrichum orbiculare is predicted to secrete hundreds of effector proteins when the pathogen infects cucurbit crops, such as cucumber and melon, and tobacco (Nicotiana benthamiana), a distantly related Solanaceae species. Here, we report the identification of sets of C. orbiculare effector genes that are differentially required for fungal virulence to two phylogenetically distant host species. Through targeted gene knockout screening of C. orbiculare ‘core’ effector candidates defined based on in planta gene expression, we identified: four host-specific virulence effectors (named effector proteins for cucurbit infection, or EPCs) that are required for full virulence of C. orbiculare to cucurbit hosts, but not to the Solanaceae host N. benthamiana; and five host-nonspecific virulence effectors, which collectively contribute to fungal virulence to both hosts. During host infection, only a small subset of genes, including the host-specific EPC effector genes, showed preferential expression on one of the hosts, while gene expression profiles of the majority of other genes, including the five host-nonspecific effector genes, were common to both hosts. This work suggests that C. orbiculare adopts a host-specific effector deployment strategy, in addition to general host-blind virulence mechanisms, for adaptation to cucurbit hosts., なぜ病原菌は特定の作物にのみ感染するのか --植物病原菌の宿主特異性の鍵因子--. 京都大学プレスリリース. 2023-03-23., Fungus has a host of issues: KyotoU discovers how toxic fungi target specific host plants. 京都大学プレスリリース. 2023-06-07.

Tomato mosaic virus (ToMV) and tobacco mosaic virus (TMV) are critical pathogens causing severe crop production losses of solanaceous plants. The present study was undertaken to evaluate the antiviral effects of extracts of Alpinia plants on ToMV and TMV infection in Nicotiana benthamiana. The aqueous extracts of Alpinia zerumbet (Pers.) B.L. Burtt and R.M. Smith and Alpinia kumatake, which grow widely in subtropical and tropical regions including East Asia, were effective in reducing ToMV infection when plants were treated prior to virus inoculation. We also found that the extracts of A. zerumbet isolated from Okinawa (Japan), locally referred to as shima-gettou, strongly inhibited ToMV and TMV infection. To obtain an active fraction, the aqueous extract of A. zerumbet isolate OG1 was separated by ethyl acetate, and the antiviral active compound was found to be present in the water layer. Based on our results, the extract of Alpinia plants has potential as an antiviral reagent for practical application in solanaceous crop production.

Proanthocyanidins (PACs) have various bioactivities, such as being anti-bacterial, anti-cancer, and anti-oxidant. Consequently, they have been vigorously studied for the development of new natural bioactive compounds. Recently, PAC was isolated from leaves and pseudostems of the medicinal plant Alpinia zerumbet (Pers.) B.L. Burtt and R.M. Smith, and it had shown in vitro antiviral activity against influenza A H1N1 viruses (IAVs). The 50% endpoint dilution method indicated that 0.1 mg/mL A. zerumbet-derived PAC (AzPAC) reduced the titer of IAVs by3 logs. The antiviral activity of AzPAC means that it can interact directly with viral particles since the antiviral activity test was done by coincubation of PAC with and IAVs before viral infection. However, few studies have investigated the preventive mechanism utilized by AzPAC on influenza virus replication. In this study, the composition of AzPAC and the affinity between AzPAC and IAVs was investigated in detail. We found that AzPAC was composed of an epicatechin, which was linked by inter-flavan bonds between the C4 and C8 positions (B2-type) and the C4 and C6 positions (B5-type) in the terminal units. A quenching assay indicated that AzPAC interacted with IAV membrane proteins, hemagglutinin and neuraminidase. Additionally, circular dichroism analysis indicated that AzPAC affected the change in the secondary structure rate of the viral membrane proteins. AzPAC was able to impair the infective process of IAVs via direct interaction with their viral membrane proteins. These results indicate that A. zerumbet is a bioresource for the development of preventive drugs against IAV infection.

Proanthocyanidins (PACs) have various bioactivities, such as being anti-bacterial, anti-cancer, and anti-oxidant. Consequently, they have been vigorously studied for the development of new natural bioactive compounds. Recently, AzPAC was isolated from the medicinal plant Alpinia zerumbet, and it was found to inhibit the infection of animal viruses, influenza A viruses (IAVs), and porcine epidemic diarrhea virus. The virucidal activity of AzPAC means that it can interact directly with viral particles. However, few studies have investigated the preventive mechanism utilized by AzPAC on influenza virus replication. In this study, the composition of AzPAC and the affinity between AzPAC and IAVs was investigated in detail. We found that AzPAC was composed of an epicatechin monomer, which was linked by inter-flavan bonds between the C4 and C8 positions (B2-type) and the C4 and C6 positions (B5-type) in the terminal units of the PAC. A quenching assay indicated that AzPAC interacted with IAV membrane proteins, hemagglutinin and neuraminidase. Additionally, circular dichroism analysis indicated that AzPAC affected the change in the secondary structure rate of the viral membrane proteins. AzPAC was able to impair the infective process of IAVs via direct interaction with their viral membrane proteins. These results indicate that A. zerumbet is an invaluable bioresource for the development of preventive drugs against IAV infection.

Phytopathogen genomes are under constant pressure to change, as pathogens are locked in an evolutionary arms race with their hosts, where pathogens evolve effector genes to manipulate their hosts, whereas the hosts evolve immune components to recognize the products of these genes. Colletotrichum higginsianum (Ch), a fungal pathogen with no known sexual morph, infects Brassicaceae plants including Arabidopsis thaliana. Previous studies revealed that Ch differs in its virulence toward various Arabidopsis thaliana ecotypes, indicating the existence of coevolutionary selective pressures. However, between-strain genomic variations in Ch have not been studied. Here, we sequenced and assembled the genome of a Ch strain, resulting in a highly contiguous genome assembly, which was compared with the chromosome-level genome assembly of another strain to identify genomic variations between strains. We found that the two closely related strains vary in terms of large-scale rearrangements, the existence of strain-specific regions, and effector candidate gene sets and that these variations are frequently associated with transposable elements (TEs). Ch has a compartmentalized genome consisting of gene-sparse, TE-dense regions with more effector candidate genes and gene-dense, TE-sparse regions harboring conserved genes. Additionally, analysis of the conservation patterns and syntenic regions of effector candidate genes indicated that the two strains vary in their effector candidate gene sets because of de novo evolution, horizontal gene transfer, or gene loss after divergence. Our results reveal mechanisms for generating genomic diversity in this asexual pathogen, which are important for understanding its adaption to hosts.

Plant pathogens secrete small proteins, known as effectors, that promote infection by manipulating host cells. Members of the phytopathogenic fungal genus Colletotrichum collectively have a broad host range and generally adopt a hemibiotrophic lifestyle that includes an initial biotrophic phase and a later necrotrophic phase. We hypothesized that Colletotrichum fungi use a set of conserved effectors during infection to support the two phases of their hemibiotrophic lifestyle. This study aimed to examine this hypothesis by identifying and characterizing conserved effectors among Colletotrichum fungi. Comparative genomic analyses using genomes of ascomycete fungi with different lifestyles identified seven effector candidates that are conserved across the genus Colletotrichum. Transient expression assays showed that one of these conserved effectors, CEC3, induces nuclear expansion and cell death in Nicotiana benthamiana, suggesting that CEC3 is involved in promoting host cell death during infection. Nuclear expansion and cell death induction were commonly observed in CEC3 homologs from four different Colletotrichum species that vary in host specificity. Thus, CEC3 proteins could represent a novel class of core effectors with functional conservation in the genus Colletotrichum.

In plants, viral diseases are second only to fungal diseases in terms of occurrence, and cause substantial damage to agricultural crops. The aqueous extracts of shell ginger, Alpinia zerumbet exhibit inhibitory effects against virus infections in belonging to the Solanaceae family. In this study, we isolated an anti-plant-virus molecule from the extracts using a conventional method involving a combination of reversed phase column chromatography, dialysis, and lyophilization. The anti-plant-virus molecule was identified as proanthocyanidin, which mostly consisted of epicatechin and exhibited more than 40 degrees of polymerization.

Members of the Colletotrichum gloeosporioides species complex are causal agents of anthracnose in many commercially important plants. Closely related strains have different levels of pathogenicity on hosts despite their close phylogenetic relationship. To gain insight into the genetics underlying these differences, we generated and annotated whole-genome assemblies of multiple isolates of C. fructicola (Cf) and C. siamense (Cs), as well as three previously unsequenced species, C. aenigma (Ca), C. tropicale and C. viniferum with different pathogenicity on strawberry. Based on comparative genomics, we identified accessory regions with a high degree of conservation in strawberry-pathogenic Cf, Cs and Ca strains. These regions encode homologs of pathogenicity-related genes known as effectors, organized in syntenic gene clusters, with copy number variations in different strains of Cf, Cs and Ca. Analysis of highly contiguous assemblies of Cf, Cs and Ca revealed the association of related accessory effector gene clusters with telomeres and repeat-rich chromosomes and provided evidence of exchange between these two genomic compartments. In addition, expression analysis indicated that orthologs in syntenic gene clusters showed a tendency for correlated gene expression during infection. These data provide insight into mechanisms by which Colletotrichum genomes evolve, acquire and organize effectors. This article is protected by copyright. All rights reserved.

Tomato mosaic virus (ToMV) and tobacco mosaic virus (TMV) are critical pathogens causing severe crop production losses of solanaceous plants. The present study was undertaken to evaluate the antiviral effects of extracts of Alpinia plants on ToMV and TMV infection in Nicotiana benthamiana. The aqueous extracts of Alpinia zerumbet (Pers.) B.L. Burtt and R.M. Smith and Alpinia kumatake, which grow widely in subtropical and tropical regions including East Asia, were effective in reducing ToMV infection when plants were treated prior to virus inoculation. We also found that the extracts of A. zerumbet isolated from Okinawa (Japan), locally referred to as shima-gettou, strongly inhibited ToMV and TMV infection. To obtain an active fraction, the aqueous extract of A. zerumbet isolate OG1 was separated by ethyl acetate, and the antiviral active compound was found to be present in the water layer. Based on our results, the extract of Alpinia plants has potential as an antiviral reagent for practical application in solanaceous crop production.

Members of theColletotrichum gloeosporioidesspecies complex are causal agents of anthracnose in a wide range of commercially important plants. To provide an in-depth overview of its diversity, we sequenced the genomes of fungi belonging to this group, including multiple strains ofC. fructicola(Cf) andC. siamense(Cs), as well as representatives of three previously unsequenced species,C. aenigma(Ca),C. tropicaleandC. viniferum. Comparisons between multipleCfandCsstrains led to the identification of accessory regions that show variable conservation in both lineages. These accessory regions encode effector candidate genes, including homologs of previously characterized effectors, organized in clusters of conserved synteny with copy number variations in different strains ofCf, CsandCa. Analysis of highly contiguous assemblies ofCf, CsandCastrains revealed the association of such accessory effector gene clusters with subtelomeric regions and repeat-rich minichromosomes and provided evidence of gene transfer between these two genomic compartments. In addition, expression analysis indicated that paralogs associated with clusters of conserved synteny showed a tendency for correlated gene expression. These data highlight the importance of subtelomeric regions and repeat-rich chromosomes to the genome plasticity ofColletotrichumfungi.

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.

Species of the fungal genus Colletotrichum are among the most devastating pathogens of agricultural crops in the world. Based on DNA sequence data (ITS, GAPDH, CHS-1, ACT, TUB2) and morphology, we revealed Colletotrichum isolates infecting the oil crop Perilla frutescens, commonly known as shiso, to represent a previously unknown species of the C. destructivum species complex and described it as C. shisoi. We found that C. shisoi appears to be able to adopt a hemibiotrophic lifestyle, characterised by the formation of biotrophic hyphae followed by severe necrotic lesions on P. frutescens, but is less virulent on Arabidopsis, compared to its close relative C. higginsianum which also belongs to the C. destructivum species complex. The genome of C. shisoi was sequenced, annotated and its predicted proteome compared with four other Colletotrichum species. The predicted proteomes of C. shisoi and C. higginsianum, share many candidate effectors, which are small, secreted proteins that may contribute to infection. Interestingly, C. destructivum species complex-specific secreted proteins showed evidence of increased diversifying selection which may be related to their host specificities.

Perception of microbe‐associated molecular patterns by host cell surface pattern recognition receptors (PRRs) triggers the intracellular activation of mitogen‐activated protein kinase (MAPK) cascades. However, it is not known how PRRs transmit immune signals to MAPK cascades in plants. Here, we identify a complete phospho‐signaling transduction pathway from PRR‐mediated pathogen recognition to MAPK activation in plants. We found that the receptor‐like cytoplasmic kinase PBL27 connects the chitin receptor complex CERK1‐LYK5 and a MAPK cascade. PBL27 interacts with both CERK1 and the MAPK kinase kinase MAPKKK5 at the plasma membrane. Knockout mutants of MAPKKK5 compromise chitin‐induced MAPK activation and disease resistance to Alternaria brassicicola . PBL27 phosphorylates MAPKKK5 in vitro , which is enhanced by phosphorylation of PBL27 by CERK1. The chitin perception induces disassociation between PBL27 and MAPKKK5 in vivo . Furthermore, genetic evidence suggests that phosphorylation of MAPKKK5 by PBL27 is essential for chitin‐induced MAPK activation in plants. These data indicate that PBL27 is the MAPKKK kinase that provides the missing link between the cell surface chitin receptor and the intracellular MAPK cascade in plants. ![][1] Chitin receptor CERK1 transmits immune signals to the intracellular MAPK cascade in plants. This occurs via phosphorylation of MAPKKK5 by the CERK1‐associated kinase PBL27, providing a missing link between pathogen perception and signaling output. [1]: /embed/graphic-1.gif

Members from Colletotrichum genus adopt a diverse range of lifestyles during infection of plants and represent a group of agriculturally devastating pathogens. In this study, we present the draft genome of Colletotrichum incanum from the spaethianum clade of Colletotrichum and the comparative analyses with five other Colletotrichum species from distinct lineages. We show that the C. incanum strain, originally isolated from Japanese daikon radish, is able to infect both eudicot plants, such as certain ecotypes of the eudicot Arabidopsis, and monocot plants, such as lily. Being closely related to Colletotrichum species both in the graminicola clade, whose members are restricted strictly to monocot hosts, and to the destructivum clade, whose members are mostly associated with dicot infections, C. incanum provides an interesting model system for comparative genomics to study how fungal pathogens adapt to monocot and dicot hosts. Genus-wide comparative genome analyses reveal that Colletotrichum species have tailored profiles of their carbohydrate-degrading enzymes according to their infection lifestyles. In addition, we show evidence that positive selection acting on secreted and nuclear localized proteins that are highly conserved may be important in adaptation to specific hosts or ecological niches.

Genomic and amino acid sequences of organisms are freely available from various public databases. We designed a genome-wide survey program, named "Ex-DOMAIN" (exhaustive domain and motif annotator using InterProScan), of protein domains and motifs to aid in the identification and characterization of proteins by using the InterProScan sequence analysis application, which can display information and annotations of targeted proteins and genes, conserved protein domains and motifs, chromosomal locations, and structural diversities of target proteins. In this study, we indicated the disease resistance genes (proteins) that play an important role in defense against pathogens in Arabidopsis thaliana (thale cress) and Cucumis sativus (cucumber), by searches based on the conserved protein domains, NB-ARC (a nucleotide-binding adaptor shared by the apoptotic protease-activating factor-1, plant resistance proteins, and Caenorhabditis elegans death-4 protein) and C-terminal leucine-rich repeat (LRR), in the nucleotide-binding domain and LRR (NLR) proteins. Our findings suggest that this program will enable searches for various protein domains and motifs in all organisms.

The hemibiotrophic pathogen Colletotrichum orbiculare preferentially expresses a necrosis and ethylene-inducing peptide 1 (Nep1)-like protein named NLP1 during the switch to necrotrophy. Here, we report that the constitutive expression of NLP1 in C. orbiculare blocks pathogen infection in multiple Cucurbitaceae cultivars via their enhanced defense responses. NLP1 has a cytotoxic activity that induces cell death in Nicotiana benthamiana. However, C. orbiculare transgenic lines constitutively expressing a mutant NLP1 lacking the cytotoxic activity still failed to infect cucumber, indicating no clear relationship between cytotoxic activity and the NLP1-dependent enhanced defense. NLP1 also possesses the microbe-associated molecular pattern (MAMP) sequence called nlp24, recognized by Arabidopsis thaliana at its central region, similar to NLPs of other pathogens. Surprisingly, inappropriate expression of a mutant NLP1 lacking the MAMP signature is also effective for blocking pathogen infection, uncoupling the infection block from the corresponding MAMP. Notably, the deletion analyses of NLP1 suggested that the C-terminal region of NLP1 is critical to enhance defense in cucumber. The expression of mCherry fused with the C-terminal 32 amino acids of NLP1 was enough to trigger the defense of cucurbits, revealing that the C-terminal region of the NLP1 protein is recognized by cucurbits and, then, terminates C. orbiculare infection.

A pair of Arabidopsis thaliana resistance proteins, RPS4 and RRS1, recognizes the cognate Avr effector from the bacterial pathogens Pseudomonas syringae pv. tomato expressing avrRps4 (Pst-avrRps4), Ralstonia solanacearum, and the fungal pathogen Colletotrichum higginsianum and leads to defense signaling activation against the pathogens. In the present study, we analyzed 14 A. thaliana accessions for natural variation in Pst-avrRps4 and C. higginsianum susceptibility, and found new compatible and incompatible Arabidopsis–pathogen interactions. We first found that A. thaliana accession Cvi-0 is susceptible to Pst-avrRps4. Interestingly, the genome sequence assembly indicated that Cvi-0 lost both RPS4 and RRS1, but not RPS4B and RRS1B, compared to the reference genome sequence from A. thaliana accession Col-0. On the other hand, the natural variation analysis of RPS4 alleles from various Arabidopsis accessions revealed that one amino-acid change, Y950H, is responsible for the loss of resistance to Pst-avrRps4 and C. higginsianum in RLD-0. Our data indicate that the amino acid change, Y950H, in RPS4 resulted in the loss of both RPS4 and RRS1 functions and resistance to pathogens.

Colletotrichum chlorophyti is a fungal pathogen that infects various herbaceous plants, including crops such as legumes, tomato, and soybean. Here, we present the genome of C. chlorophyti NTL11, isolated from tomato. Analysis of this genome will allow a clearer understanding of the molecular mechanisms underlying fungal host range and pathogenicity.

Recognition of microbe-associated molecular patterns (MAMPs) initiates pattern-triggered immunity in host plants. Pattern recognition receptors (PRRs) and receptor-like cytoplasmic kinases (RLCKs) are the major components required for sensing and transduction of these molecular patterns. However, the regulation of RLCKs by PRRs and their specificity remain obscure. In this study we show that PBL27, an Arabidopsis ortholog of OsRLCK185, is an immediate downstream component of the chitin receptor CERK1 and contributes to the regulation of chitin-induced immunity in Arabidopsis. Knockout of PBL27 resulted in the suppression of several chitin-induced defense responses, including the activation of MPK3/6 and callose deposition as well as in disease resistance against fungal and bacterial infections. On the other hand, the contribution of PBL27 to flg22 signaling appears to be very limited, suggesting that PBL27 selectively regulates defense signaling downstream of specific PRR complexes. In vitro phosphorylation experiments showed that CERK1 preferentially phosphorylated PBL27 in comparison to BIK1, whereas phosphorylation of PBL27 by BAK1 was very low compared with that of BIK1. Thus, the substrate specificity of the signaling receptor-like kinases, CERK1 and BAK1, may determine the preference of downstream RLCKs.

Arabidopsis thaliana leucine-rich repeat-containing (NLR) proteins RPS4 and RRS1, known as dual resistance proteins, confer resistance to multiple pathogen isolates, such as the bacterial pathogens Pseudomonas syringae and Ralstonia solanacearum and the fungal pathogen Colletotrichum higginsianum. RPS4 is a typical Toll/interleukin 1 Receptor (TIR)-type NLR, whereas RRS1 is an atypical TIR-NLR that contains a leucine zipper (LZ) motif and a C-terminal WRKY domain. RPS4 and RRS1 are localised near each other in a head-to-head orientation. In this study, direct mutagenesis of the C-terminal LZ motif in RRS1 caused an autoimmune response and stunting in the mutant. Co-immunoprecipitation analysis indicated that full-length RPS4 and RRS1 are physically associated with one another. Furthermore, virus-induced gene silencing experiments showed that hypersensitive-like cell death triggered by RPS4/LZ motif-mutated RRS1 depends on EDS1. In conclusion, we suggest that the RRS1-LZ motif is crucial for the regulation of the RPS4/RRS1 complex.

To develop a model system for studying host tree–fungal pathogen interactions, we focused on Populus as a model tree and the fungus Colletotrichum gloeosporioides as a model pathogen. Although hybrid Populus is currently planted worldwide for biomass production and other purposes, the hybrid often lacks the tolerance of native species to cold, drought, and disease. Populus diseases have increased with the rapid development of Populus plantations worldwide. To investigate the susceptibility of hybrid aspen plants to the fungal pathogen Colletotrichum, which has a wide range of hosts, hybrid aspen plants were inoculated with Colletotrichum species such as C. dematium, C. destructivum, C. gloeosporioides, C. higginsianum, and C. orbiculare. C. gloeosporioides strains isolated from strawberry plants could infect hybrid aspen. These results indicate that monoculture of hybrid Populus may be potentially susceptible to local pathogens. The experimental system using hybrid aspen and C. gloeosporioides presented here will bring a lot of fruitful information, as first model system of host tree–fungal pathogen interactions.

Arabidopsis belongs to the Brassicaceae family and plays an important role as a model plant for which researchers have developed fine-tuned genome resources. Genome sequencing projects have been initiated for other members of the Brassicaceae family. Among these projects, research on Chinese cabbage (Brassica rapa subsp. pekinensis) started early because of strong interest in this species. Here, we report the development of a library of Chinese cabbage full-length cDNA clones, the RIKEN BRC B. rapa full-length cDNA (BBRAF) resource, to accelerate research on Brassica species. We sequenced 10 000 BBRAF clones and confirmed 5476 independent clones. Most of these cDNAs showed high homology to Arabidopsis genes, but we also obtained more than 200 cDNA clones that lacked any sequence homology to Arabidopsis genes. We also successfully identified several possible candidate marker genes for plant defence responses from our analysis of the expression of the Brassica counterparts of Arabidopsis marker genes in response to salicylic acid and jasmonic acid. We compared gene expression of these markers in several Chinese cabbage cultivars. Our BBRAF cDNA resource will be publicly available from the RIKEN Bioresource Center and will help researchers to transfer Arabidopsis-related knowledge to Brassica crops.

Pseudomonas, Ralstonia, and Xanthomonas are important genera of plant pathogenic bacteria that affect commercially cultivated crops. We developed an assay to assess the viability and development of bacterial pathogens in Arabidopsis thaliana by detecting rpoD mRNA using a quantitative reverse transcription-polymerase chain reaction (qRT-PCR). rpoD is a representative housekeeping gene that encodes the principal RNA polymerase sigma factor. Quantification of rpoD mRNA by qRT-PCR allowed easy detection of viability and development of the pathogens in infected plant tissue. Therefore, qRT-PCR technology provides new research opportunities for investigation of host–pathogen interactions.

The Brassicaceae family which includes Arabidopsis thaliana, is a natural priority for reaching beyond botanical models to more deeply sample angiosperm genomic and functional diversity. Here we report the draft genome sequence and its annoation of Brassica rapa, one of the two ancestral species of oilseed rape. We modeled 41,174 protein-coding genes in the B. rapa genome. B. rapa has experienced only the second genome triplication reported to date, with its close relationship to A. thaliana providing a useful outgroup for investigating many consequences of triplication for its structural and functional evolution. The extent of gene loss (fractionation) among triplicated genome segments varies, with one copy containing a greater proportion of genes expected to have been present in its ancestor (70 percent) than the remaining two (46 percent and 36 percent). Both a generally rapid evolutionary rate, and specific copy number amplifications of particular gene families, may contribute to the remarkable propensity of Brassica species for the development of new morphological variants. The B. rapa genome provides a new resource for comparative and evolutionary analysis of the Brassicaceae genomes and also a platform for genetic improvement of Brassica oil and vegetable crops.

In the food industry, glutamate fermentation by-product (GFB) is generated by purifying glutamate products from microbial fermentation. The potential applications of GFB for upgrading agricultural soil, for foliar fertility, and as plant plankton for shrimp have been studied. We examined the efficacy of GFB foliar application and determined that GFB treatment increased the resistance of Arabidopsis leaves to infection by bacterial pathogens. Microarray gene expression analysis of Arabidopsis leaves after treatment with GFB indicated that the expression of plant defence-related genes increased. In Corynebacterium fermentation, the active substances for induction of the defence response were extracted or solubilized after treatment with heating under acidic conditions. This extract was also effective in strawberry and grape leaves for the induction of hydrogen peroxide production. These findings suggest that foliar application of GFB that contains elicitor molecules derived from fermentation bacteria is useful for plant protection in agricultural fields.

Colletotrichum higginsianum causes typical anthracnose lesions on a wide range of cruciferous plants. Currently, assays for C. higginsianum infection of host plants rely on visual diagnosis of symptoms. In this study, we developed an assay to estimate the viability and development of C. higginsianum in Arabidopsis thaliana and Brassica rapa by detecting C. higginsianum-actin (Ch-ACT) mRNA using quantitative reverse transcription-PCR. Ch-ACT mRNA was specifically targeted because of its constitutive expression throughout the cell cycle. The methodology described will be useful for assessing potential antifungal compounds and breeding disease-resistant crops.

Transcription factors of the DRE-Binding1 (DREB1)/C-repeat binding factor family specifically interact with a cis-acting dehydration-responsive element/C-repeat involved in low-temperature stress-responsive gene expression in Arabidopsis (Arabidopsis thaliana). Expression of DREB1s is induced by low temperatures and is regulated by the circadian clock under unstressed conditions. Promoter sequences of DREB1s contain six conserved motifs, boxes I to VI. We analyzed the promoter region of DREB1C using transgenic plants and found that box V with the G-box sequence negatively regulates DREB1C expression under circadian control. The region around box VI contains positive regulatory elements for low-temperature-induced expression of DREB1C. Using yeast one-hybrid screens, we isolated cDNA encoding the transcriptional factor Phytochrome-Interacting Factor7 (PIF7), which specifically binds to the G-box of the DREB1C promoter. The PIF7 gene was expressed in rosette leaves, and the PIF7 protein was localized in the nuclei of the cells. Transactivation experiments using Arabidopsis protoplasts indicated that PIF7 functions as a transcriptional repressor for DREB1C expression and that its activity is regulated by PIF7-interacting factors TIMING OF CAB EXPRESSION1 and Phytochrome B, which are components of the circadian oscillator and the red light photoreceptor, respectively. Moreover, in the pif7 mutant, expression of DREB1B and DREB1C was not repressed under light conditions, indicating that PIF7 functions as a transcriptional repressor for the expression of DREB1B and DREB1C under circadian control. This negative regulation of DREB1 expression may be important for avoiding plant growth retardation by the accumulation of DREB1 proteins under unstressed conditions.

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.

ABA and salicylic acid (SA) are believed to act antagonistically. We previously reported that an ABA-hypersensitive mutant ahg2-1, which had reduced expression of poly(A)-specific ribonuclease (PARN), exhibited pleiotropic phenotypes including unique enhanced ABA- and SA-sensitive phenotypes. In this study, we characterized the increased SA-sensitive phenotype of this mutant in detail and addressed its relationship with ABA-related and dwarf phenotypes. We found that the ahg2-1 mutant had a high endogenous SA level and an elevated resistance to bacterial pathogens. Double mutant analyses showed that Arabidopsis plants defective in the SA signaling pathway (npr1 and pad4 mutants and nahG transgenic plants) could suppress neither the ABA hypersensitivity nor the dwarf phenotypes. These results indicate that ABA-related, SA-related and dwarf phenotypes of the ahg2-1 mutant are independent of each other. To obtain more insight into the molecular basis of the effect of ahg2-1, microarray analyses were conducted not only for ahg2-1 but also for ahg2sid2 or ahg2abi1 so as to reduce the secondary effects of SA or ABA. The resulting data indicate that ahg2-1 has a unique gene expression profile, consistent with the novel phenotype of this mutant. Detailed comparison of the expression profiles of up- or down-regulated genes implied that ahg2-1 somehow affects mitochondrial function. Our data suggest that a partial loss of PARN activity affects ABA, SA and mitochondrial function independently, and that the regulation of mRNA levels is deeply implicated in diverse cellular functions.

Colletotrichum higginsianum is a hemibiotrophic fungal pathogen that causes anthracnose disease on Arabidopsis and other crucifer hosts. By exploiting natural variation in Arabidopsis we identified a resistance locus that is shared by four geographically distinct accessions (Ws-0, Kondara, Gifu-2 and Can-0). A combination of quantitative trait loci (QTL) and Mendelian mapping positioned this locus within the major recognition gene complex MRC-J on chromosome 5 containing the Toll-interleukin-1 receptor/nucleotide-binding site/leucine-rich repeat (TIR-NB-LRR) genes RPS4 and RRS1 that confer dual resistance to C. higginsianum in Ws-0 (Narusaka et al., 2009). We find that the resistance shared by these diverse Arabidopsis accessions is expressed at an early stage of fungal invasion, at the level of appressorial penetration and establishment of intracellular biotrophic hyphae, and that this determines disease progression. Resistance is not associated with host hypersensitive cell death, an oxidative burst or callose deposition in epidermal cells but requires the defense regulator EDS1, highlighting new functions of TIR-NB-LRR genes and EDS1 in limiting early establishment of fungal biotrophy. While the Arabidopsis accession Ler-0 is fully susceptible to C. higginsianum infection, Col-0 displays intermediate resistance that also maps to MRC-J. By analysis of null mutants of RPS4 and RRS1 in Col-0 we show that these genes, individually, do not contribute strongly to C. higginsianum resistance but are both required for resistance to Pseudomonas syringae bacteria expressing the Type III effector, AvrRps4. We conclude that distinct allelic forms of RPS4 and RRS1 probably cooperate to confer resistance to different pathogens.

To develop a screening system for plant defense activators, which are novel substances that protect plants by enhancing their inherent disease-resistance mechanisms, we utilized a GUS histochemical staining assay using promoters of the defense-related genes, PR-1 and PDF1.2. We can perform about 1,000 screenings per week per person by this high- throughput screening method. This GUS assay for plant defense activator candidates was evaluated by QRT-PCR analysis to elucidate the functions of the plant defense activators in detail. In the present preliminary screening, we evaluated two hundred chemicals chosen at random. Some chemicals induced GUS activity in a PR-1 promoter::GUS transformant, i.e., abietic acid, allose, glycine, and thymol. The induction of PR-1 expression by the treatments with these chemicals was confirmed using QRT-PCR. The foliar treatment with abietic acid 1 d prior to inoculation with the fungal pathogen Colletotrichum higginsianum led to a significant reduction of necrotic surface area compared with distilled water treated controls, as observed 6 d after inoculation. These results suggest that this GUS histochemical staining assay is an effective and available screening system for plant defense activators.

SigA-binding protein (SIB A) is a nuclear-encoded chloroplast-targeted protein that interacts with the plastid-encoded plastid RNA polymerase σ-factor SigA (Sig1). In this study, the SIB A gene responded rapidly to salicylic acid (SA) treatment, but responded slowly to ethylene (ET) and jasmonic acid (JA) treatments as determined with microarray and quantitative real-time RT-PCR analyses. Expression of the SIB A gene increased rapidly, with a peak at 2 h after SA treatment and then decreased gradually. In contrast, expression of the PR-1 gene, a marker gene on the SA defense signaling pathway, increased gradually between 0 and 24 h after SA treatment. In addition, transcription levels of SIB A and PR-1 decreased between 0 and 24 h in the benzothiadiazole (BTH)-treated npr1-1 plants. We suggest that the SIB A gene is downstream of NPR1 on the SA signaling pathway. From the results of our analysis of the 1.5-kbp promoter region of the SIB A gene in Arabidopsis, the isolated promoter region of the SIB A gene seems able to drive the transcription of the GUS gene to elevated levels more rapidly in transgenic Arabidopsis plants undergoing SA treatment compared with levels of PR-1. To analyze biological function of SIB A and investigate SIB A-regulated genes, we generated transgenic Arabidopsis plants in which SIB A was overexpressed. As a result, ROS-related genes, such as glutathione transferase, were up-regulated in plants overexpressing SIB A. However, SIB A-overexpressing plants were not resistant to Pseudomonas syringae pv. tomato DC3000 strain. The SIB A gene has promise as a new tool to analyze the SA-signaling pathway and the role of the chloroplast in plant defense responses.

Chitin is a major component of fungal cell walls and serves as a microbe-associated molecular pattern (MAMP) for the detection of various potential pathogens in innate immune systems of both plants and animals. We recently showed that chitin elicitor-binding protein (CEBiP), plasma membrane glycoprotein with LysM motifs, functions as a cell surface receptor for chitin elicitor in rice. The predicted structure of CEBiP does not contain any intracellular domains, suggesting that an additional component(s) is required for signaling through the plasma membrane into the cytoplasm. Here, we identified a receptor-like kinase, designated CERK1, which is essential for chitin elicitor signaling in Arabidopsis . The KO mutants for CERK1 completely lost the ability to respond to the chitin elicitor, including MAPK activation, reactive oxygen species generation, and gene expression. Disease resistance of the KO mutant against an incompatible fungus, Alternaria brassicicola , was partly impaired. Complementation with the WT CERK1 gene showed cerk1 mutations were responsible for the mutant phenotypes. CERK1 is a plasma membrane protein containing three LysM motifs in the extracellular domain and an intracellular Ser/Thr kinase domain with autophosphorylation/myelin basic protein kinase activity, suggesting that CERK1 plays a critical role in fungal MAMP perception in plants.

LjABCG1, a full-size ABCG subfamily of ATP-binding cassette proteins of a model legume, Lotus japonicus, was reported as a gene highly expressed during the early stages of nodulation, but have not been characterized in detail. In this study we showed that the induction of LjABCG1 expression was remarkable by methyl jasmonate treatment, and reporter gene experiments indicated that LjABCG1 was strongly expressed in the nodule parenchyma and cell layers adjacent to the root vascular tissue toward the nodule. LjABCG1 was suggested to be localized at the plasma membrane based on the fractionation of microsomal membranes as well as separation via aqueous two-phase partitioning. The physiological functions of LjABCG1 in symbiosis and pathogenesis were analyzed in homologous and heterologous systems. LjABCG1 knock-down L. japonicus plants did not show clear phenotypic differences in nodule formation, and not in defense against Pseudomonas syringae, either. In contrast, when LjABCG1 was expressed in the Arabidopsis pdr8-1 mutant, the penetration frequency of Phytophthora infestans, a potato late blight pathogen, was significantly reduced in LjABCG1/pdr8-1 than in pdr8-1 plants. This finding indicated that LjABCG1, at least partially, complemented the phenotype of pdr8 in Arabidopsis, suggesting the multiple roles of this protein in plant-microbe interactions.

Bacterial wilt phytopathogen Ralstonia solanacearum is a serious soil-borne disease that attacks several economically important plants worldwide, including Brassicaceae. Previous studies indicate that recognition of avirulence (Avr)-effector PopP2 by resistance (R) protein, RRS1-R, and physical interaction between RRS1-R and PopP2 in the nucleus are required for resistance. Of late, we showed that a pair of Arabidopsis thaliana TIR-NLR proteins, RRS1 and RPS4, function together in disease resistance against multiple pathogen isolates. Here, we report that dual R proteins, RRS1 and RPS4, from A. thaliana ecotype Wassilewskija confer resistance to bacterial wilt in transgenic Brassica crops. For practical applications, this finding may provide a new strategy for developing disease resistant plants that express R genes from other plants.

Housaku Monogatari (HM) is a plant activator prepared from a yeast cell wall extract. We examined the efficacy of HM application and observed that HM treatment increased the resistance of Arabidopsis thaliana and Brassica rapa leaves to bacterial and fungal infections. HM reduced the severity of bacterial leaf spot and anthracnose on A. thaliana and Brassica crop leaves with protective effects. In addition, gene expression analysis of A. thaliana plants after treatment with HM indicated increased expression of several plant defense-related genes. HM treatment appears to induce early activation of jasmonate/ethylene and late activation of salicylic acid (SA) pathways. Analysis using signaling mutants revealed that HM required SA accumulation and SA signaling to facilitate resistance to the bacterial pathogen Pseudomonas syringae pv. maculicola and the fungal pathogen Colletotrichum higginsianum. In addition, HM-induced resistance conferred chitin-independent disease resistance to bacterial pathogens in A. thaliana. These results suggest that HM contains multiple microbe-associated molecular patterns that activate defense responses in plants. These findings suggest that the application of HM is a useful tool that may facilitate new disease control methods.

To perform comparative sequence and transcriptome analyses between Brassica rapa and Arabidopsis thaliana, we prepared a B. rapa cDNA microarray using 1,820 (ca. 2 K) cDNA clones selected from 2,166 non-redundant sequences of cDNA library of Chinese cabbage. The gene expression during infection with fungal pathogen Colletotrichum higginsianum and treatments with signaling molecules was analyzed using 2 K B. rapa and 1.2 K Arabidopsis cDNA microarrays. In B. rapa, the results suggested a large correlation coefficient between compatible pathogen C. higginsianum-infection and the treatment with salicylic acid, methyl jasmonate, or ethephon. The expression profiles of 145 counterpart gene sets between the B. rapa and Arabidopsis were distributed in the self-organizing map analysis. The 28% of them indicated similarities in the two species transcriptome. These expressed sequence tag (EST) and microarray data should provide a valuable resource for functional genomics on the crops.