Map-Based Cloning and Characterization of a Major QTL Gene, FfR1, Which Confers Resistance to Rice Bakanae Disease.

Bakanae disease (BD), caused by the fungal pathogen Fusarium fujikuroi, is a serious threat to rice production worldwide. Breeding elite rice varieties resistant to BD requires the identification of resistance genes. Previously, we discovered a resistant quantitative trait locus (QTL), qFfR1, in a Korean japonica rice variety, Nampyeong. In this study, we fine-mapped qFfR1 with a Junam*4/Nampyeong BC3F3 population and delimited its location to a 37.1 kb region on chromosome 1. Complementation experiments with seven candidate genes in this region revealed that OsI_02728 is the gene for qFfR1. This gene encodes a protein with a typical leucine-rich repeat (LRR) receptor-like protein structure. RNA-sequencing-based transcriptomic analysis revealed that FfR1 induces the transcription of defense genes, including lignin and terpenoid biosynthesis genes, pathogenesis-related genes, and thionin genes. These results may facilitate investigations into the molecular mechanisms underlying BD resistance, including molecular patterns of Fusarium fujikuroi interacting with FfR1 and players working in signal transduction pathways downstream of FfR1, and the breeding of new BD-resistant varieties by providing a BD resistance gene with its precise selection marker. This will contribute to efficient control of BD, which is becoming more prevalent according to temperature rises due to climate change.

Comparative genomics of geographically distant Fusarium fujikuroi isolates revealed two distinct pathotypes correlating with secondary metabolite profiles.

Fusarium fujikuroi causes bakanae ("foolish seedling") disease of rice which is characterized by hyper-elongation of seedlings resulting from production of gibberellic acids (GAs) by the fungus. This plant pathogen is also known for production of harmful mycotoxins, such as fusarins, fusaric acid, apicidin F and beauvericin. Recently, we generated the first de novo genome sequence of F. fujikuroi strain IMI 58289 combined with extensive transcriptional, epigenetic, proteomic and chemical product analyses. GA production was shown to provide a selective advantage during infection of the preferred host plant rice. Here, we provide genome sequences of eight additional F. fujikuroi isolates from distant geographic regions. The isolates differ in the size of chromosomes, most likely due to variability of subtelomeric regions, the type of asexual spores (microconidia and/or macroconidia), and the number and expression of secondary metabolite gene clusters. Whilst most of the isolates caused the typical bakanae symptoms, one isolate, B14, caused stunting and early withering of infected seedlings. In contrast to the other isolates, B14 produced no GAs but high amounts of fumonisins during infection on rice. Furthermore, it differed from the other isolates by the presence of three additional polyketide synthase (PKS) genes (PKS40, PKS43, PKS51) and the absence of the F. fujikuroi-specific apicidin F (NRPS31) gene cluster. Analysis of additional field isolates confirmed the strong correlation between the pathotype (bakanae or stunting/withering), and the ability to produce either GAs or fumonisins. Deletion of the fumonisin and fusaric acid-specific PKS genes in B14 reduced the stunting/withering symptoms, whereas deletion of the PKS51 gene resulted in elevated symptom development. Phylogenetic analyses revealed two subclades of F. fujikuroi strains according to their pathotype and secondary metabolite profiles.

Proteomics of rice and Cochliobolus miyabeanus fungal interaction: insight into proteins at intracellular and extracellular spaces.

Necrotrophic fungal pathogen Cochliobolus miyabeanus causes brown spot disease in rice leaves upon infection, resulting in critical rice yield loss. To better understand the rice-C. miyabeanus interaction, we employed proteomic approaches to establish differential proteomes of total and secreted proteins from the inoculated leaves. The 2DE approach after PEG-fractionation of total proteins coupled with MS (MALDI-TOF/TOF and nESI-LC-MS/MS) analyses led to identification of 49 unique proteins out of 63 differential spots. SDS-PAGE in combination with nESI-LC-MS/MS shotgun approach was applied to identify secreted proteins in the leaf apoplast upon infection and resulted in cataloging of 501 unique proteins, of which 470 and 31 proteins were secreted from rice and C. miyabeanus, respectively. Proteins mapped onto metabolic pathways implied their reprogramming upon infection. The enzymes involved in Calvin cycle and glycolysis decreased in their protein abundance, whereas enzymes in the TCA cycle, amino acids, and ethylene biosynthesis increased. Differential proteomes also generated distribution of identified proteins in the intracellular and extracellular spaces, providing a better insight into defense responses of proteins in rice against C. miyabeanus. Established proteome of the rice-C. miyabeanus interaction serves not only as a good resource for the scientific community but also highlights its significance from biological aspects.

Quantification of Alternaria brassicicola infection in the Arabidopsis thaliana and Brassica rapa subsp. pekinensis.

Black spot caused by Alternaria brassicicola is an important fungal disease affecting cruciferous crops, including Korean cabbage (Brassica rapa subsp. pekinensis). The interaction between Arabidopsis thaliana and Alt. brassicicola is a representative model system, and objective estimation of disease progression is indispensable for accurate functional analyses. Five strains caused black spot symptom progression on Korean cabbage and Ara. thaliana ecotype Col-0. In particular, challenge with the strains Ab44877 and Ab44414 induced severe black spot progression on Korean cabbage. Ab44877 was also highly infective on Col-0; however, the virulence of Ab44414 and the remaining strains on Col-0 was lower. To unveil the relationship between mycelial growth in the infected tissues and symptom progression, we have established a reliable quantification method using real-time PCR that employs a primer pair and dual-labelled probe specific to a unigene encoding A. brassicicola SCYTALONE DEHYDRATASE1 (AbSCD1), which is involved in fungal melanin biosynthesis. Plotting the crossing point values from the infected tissue DNA on a standard curve revealed active fungal ramification of Ab44877 in both host species. In contrast, the proliferation rate of Ab44414 in Korean cabbage was 3.8 times lower than that of Ab44877. Massive infective mycelial growth of Ab44877 was evident in Col-0; however, inoculation with Ab44414 triggered epiphytic growth rather than actual in planta ramification. Mycelial growth did not always coincide with symptom development. Our quantitative evaluation system is applicable and reliable for the objective estimation of black spot disease severity.

The U-Box/ARM E3 ligase PUB13 regulates cell death, defense, and flowering time in Arabidopsis.

The components in plant signal transduction pathways are intertwined and affect each other to coordinate plant growth, development, and defenses to stresses. The role of ubiquitination in connecting these pathways, particularly plant innate immunity and flowering, is largely unknown. Here, we report the dual roles for the Arabidopsis (Arabidopsis thaliana) Plant U-box protein13 (PUB13) in defense and flowering time control. In vitro ubiquitination assays indicated that PUB13 is an active E3 ubiquitin ligase and that the intact U-box domain is required for the E3 ligase activity. Disruption of the PUB13 gene by T-DNA insertion results in spontaneous cell death, the accumulation of hydrogen peroxide and salicylic acid (SA), and elevated resistance to biotrophic pathogens but increased susceptibility to necrotrophic pathogens. The cell death, hydrogen peroxide accumulation, and resistance to necrotrophic pathogens in pub13 are enhanced when plants are pretreated with high humidity. Importantly, pub13 also shows early flowering under middle- and long-day conditions, in which the expression of SUPPRESSOR OF OVEREXPRESSION OF CONSTANS1 and FLOWERING LOCUS T is induced while FLOWERING LOCUS C expression is suppressed. Finally, we found that two components involved in the SA-mediated signaling pathway, SID2 and PAD4, are required for the defense and flowering-time phenotypes caused by the loss of function of PUB13. Taken together, our data demonstrate that PUB13 acts as an important node connecting SA-dependent defense signaling and flowering time regulation in Arabidopsis.

Complete Genome Sequence of Ralstonia solanacearum WR-1, a Virulent Strain on Solanum lycopersicum.

Ralstonia solanacearum is a bacterial wilt pathogen of Solanum lycopersicum. Its pathogenicity is the result of coevolution during continuous interaction with its host plants under given biotic and abiotic environments. To elucidate clues for pathogenicity of our WR-1 strain, its genome sequence was analyzed.

Complete Genome Sequence of Ralstonia solanacearum Strain Bs715, a Member of Biovar 4 and a Strong Pathogen of Bacterial Wilt on Solanum lycopersicum.

Ralstonia solanacearum is a notorious pathogen of bacterial wilt on Solanum lycopersicum. Most isolates from diseased tomato tissues are biovar 3, and their genomes are publicly available; however, information on biovar 4 strains is limited. Here, the complete genome sequence of R. solanacearum Bs715, a biovar 4 strain, is presented.

Complete Genome Sequence of Ralstonia pseudosolanacearum Strain Sw698, Isolated from Solanum lycopersicum.

Ralstonia pseudosolanacearum is a member of the Ralstonia solanacearum species complex (RSSC), which is composed of three species and diverse subspecific groups. Some strains cause bacterial wilt in Solanum lycopersicum; others are beneficial for their hosts. Herein, we present the complete genome sequence of an RSSC strain, Sw698, beneficial for S. lycopersicum growth.

Inhibitor of apoptosis (IAP)-like protein lacks a baculovirus IAP repeat (BIR) domain and attenuates cell death in plant and animal systems.

A novel Arabidopsis thaliana inhibitor of apoptosis was identified by sequence homology to other known inhibitor of apoptosis (IAP) proteins. Arabidopsis IAP-like protein (AtILP) contained a C-terminal RING finger domain but lacked a baculovirus IAP repeat (BIR) domain, which is essential for anti-apoptotic activity in other IAP family members. The expression of AtILP in HeLa cells conferred resistance against tumor necrosis factor (TNF)-α/ActD-induced apoptosis through the inactivation of caspase activity. In contrast to the C-terminal RING domain of AtILP, which did not inhibit the activity of caspase-3, the N-terminal region, despite displaying no homology to known BIR domains, potently inhibited the activity of caspase-3 in vitro and blocked TNF-α/ActD-induced apoptosis. The anti-apoptotic activity of the AtILP N-terminal domain observed in plants was reproduced in an animal system. Transgenic Arabidopsis lines overexpressing AtILP exhibited anti-apoptotic activity when challenged with the fungal toxin fumonisin B1, an agent that induces apoptosis-like cell death in plants. In AtIPL transgenic plants, suppression of cell death was accompanied by inhibition of caspase activation and DNA fragmentation. Overexpression of AtILP also attenuated effector protein-induced cell death and increased the growth of an avirulent bacterial pathogen. The current results demonstrated the existence of a novel plant IAP-like protein that prevents caspase activation in Arabidopsis and showed that a plant anti-apoptosis gene functions similarly in plant and animal systems.

A rice orthologue of the ABA receptor, OsPYL/RCAR5, is a positive regulator of the ABA signal transduction pathway in seed germination and early seedling growth.

Abscisic acid (ABA) is a phytohormone that positively regulates seed dormancy and stress tolerance. PYL/RCARs were identified an intracellular ABA receptors regulating ABA-dependent gene expression in Arabidopsis thaliana. However, their function in monocot species has not been characterized yet. Herein, it is demonstrated that PYL/RCAR orthologues in Oryza sativa function as a positive regulator of the ABA signal transduction pathway. Transgenic rice plants expressing OsPYL/RCAR5, a PYL/RCAR orthologue of rice, were found to be hypersensitive to ABA during seed germination and early seedling growth. A rice ABA signalling unit composed of OsPYL/RCAR5, OsPP2C30, SAPK2, and OREB1 for ABA-dependent gene regulation was further identified, via interaction assays and a transient gene expression assay. Thus, a core signalling unit for ABA-responsive gene expression modulating seed germination and early seedling growth in rice has been unravelled. This study provides substantial contributions toward understanding the ABA signal transduction pathway in rice.

Rhizobacteria-induced priming in Arabidopsis is dependent on ethylene, jasmonic acid, and NPR1.

A nonpathogenic rhizobacterium, Pseudomonas putida LSW17S, elicited systemic protection against Fusarium wilt and pith necrosis caused by Fusarium oxysporum f. sp. lycopersici and P. corrugata in tomato (Lycopersicon esculentum L.). LSW17S also confers disease resistance against P. syringae pv. tomato DC3000 (DC3000) on Arabidopsis ecotype Col-0. To investigate mechanisms underlying disease protection, expression patterns of defense-related genes PR1, PR2, PR5, and PDF1.2 and cellular defense responses such as hydrogen peroxide accumulation and callose deposition were investigated. LSW17S treatment exhibited the typical phenomena of priming. Strong and faster transcription of defense-related genes was induced and hydrogen peroxide or callose were accumulated in Arabidopsis treated with LSW17S and infected with DC3000. In contrast, individual actions of LSW17S and DC3000 did not elicit rapid molecular and cellular defense responses. Priming by LSW17S was translocated systemically and retained for more than 10 days. Treatment with LSW17S reduced pathogen proliferation in Arabidopsis ecotype Col-0 expressing bacterial NahG; however, npr1, etr1, and jar1 mutations impaired inhibition of pathogen growth. Cellular and molecular priming responses support these results. In sum, LSW17S primes Arabidopsis for NPR1-, ethylene-, and jasmonic acid-dependent disease resistance, and efficient molecular and cellular defense responses.

Calcium Restores Prepenetration Morphogenesis Abolished by Methylglyoxal-Bis-Guanyl Hydrazone in Cochliobolus miyabeanus Infecting Rice.

ABSTRACT Cochliobolus miyabeanus forms a specialized infection structure, an appressorium, to infect its host rice plants. Curtailment of prepenetration development by spermidine and spermine was more evident in appressorium development and germination remained unaffected, whereas putrescine and methylglyoxal-bis-guanyl hydrazone (MGBG) impaired both morphogenetic events. Exogenous calcium nullified the inhibitory effect of MGBG on the prepenetration development in vitro and in vivo and the disease progression. High levels of polyamines were detected in freshly collected conidia, but the amounts were reduced during germination and appressorium formation. MGBG fortified the decrease of polyamines within conidia under development and calcium amendment did not affect the reduction. Hard-surface contact augmented messenger RNA synthesis of calmodulin gene (CmCaM) and protein kinase C (PKC) activity in germinating or appressorium-forming conidia. Calcium restored transcription of CmCaM and upregulation of PKC activity suppressed by MGBG. Taken together, fine-tuning of intracellular polyamine transition is indispensable for the conidial germination and appressorium formation in C. miyabeanus. Biochemical and molecular analyses revealed that the MGBG-acting site or sites are upstream of Ca(2+)-dependent signaling pathways regulating prepenetration morphogenesis of C. miyabeanus causing rice brown leaf spot.

Multi-Homologous Recombination-Based Gene Manipulation in the Rice Pathogen Fusarium fujikuroi.

Gene disruption by homologous recombination is widely used to investigate and analyze the function of genes in Fusarium fujikuroi, a fungus that causes bakanae disease and root rot symptoms in rice. To generate gene deletion constructs, the use of conventional cloning methods, which rely on restriction enzymes and ligases, has had limited success due to a lack of unique restriction enzyme sites. Although strategies that avoid the use of restriction enzymes have been employed to overcome this issue, these methods require complicated PCR steps or are frequently inefficient. Here, we introduce a cloning system that utilizes multi-fragment assembly by In-Fusion to generate a gene disruption construct. This method utilizes DNA fragment fusion and requires only one PCR step and one reaction for construction. Using this strategy, a gene disruption construct for Fusarium cyclin C1 (FCC1 ), which is associated with fumonisin B1 biosynthesis, was successfully created and used for fungal transformation. In vivo and in vitro experiments using confirmed fcc1 mutants suggest that fumonisin production is closely related to disease symptoms exhibited by F. fujikuroi strain B14. Taken together, this multi-fragment assembly method represents a simpler and a more convenient process for targeted gene disruption in fungi.

Proteome Analysis of Disease Resistance against Ralstonia solanacearum in Potato Cultivar CT206-10.

Potato is one of the most important crops worldwide. Its commercial cultivars are highly susceptible to many fungal and bacterial diseases. Among these, bacterial wilt caused by Ralstonia solanacearum causes significant yield loss. In the present study, integrated proteomics and genomics approaches were used in order to identify bacterial wilt resistant genes from Rs resistance potato cultivar CT-206-10. 2-DE and MALDI-TOF/TOF-MS analysis identified eight differentially abundant proteins including glycine-rich RNA binding protein (GRP), tomato stress induced-1 (TSI-1) protein, pathogenesis-related (STH-2) protein and pentatricopeptide repeat containing (PPR) protein in response to Rs infection. Further, semi-quantitative RT-PCR identified up-regulation in transcript levels of all these genes upon Rs infection. Taken together, our results showed the involvement of the identified proteins in the Rs stress tolerance in potato. In the future, it would be interesting to raise the transgenic plants to further validate their involvement in resistance against Rs in potato.

Rice Defense Mechanisms Against Cochliobolus miyabeanus and Magnaporthe grisea Are Distinct.

ABSTRACT Responses of rice to Magnaporthe grisea and Cochliobolus miyabeanus were compared. In Tetep, a rice cultivar resistant to both fungi, pathogen inoculation rapidly triggered the hypersensitive reaction (HR), resulting in microscopic cell death. In rice cv. Nakdong, susceptible to both pathogens, M. grisea did not cause HR, whereas C. miyabeanus caused rapid cell death similar to that associated with HR, which appeared similar to that observed in cv. Tetep, yet failed to block fungal ramification. Treatment with conidial germination fluid (CGF) from C. miyabeanus induced rapid cell death in both cultivars, suggesting the presence of phytotoxins in CGF. Pretreatment of cv. Nakdong with CGF significantly increased resistance to M. grisea, while the same treatment was ineffective against C. miyabeanus. Similarly, in cv. Nakdong, benzothiadiazole (BTH) significantly increased resistance to M. grisea, but was ineffective against C. miyabeanus. Methyl jasmonate (MeJA) treatment appeared to be ineffective against either fungus. Increased resistance of cv. Nakdong to M. grisea by BTH or CCF treatment was correlated with more rapid induction of three monitored PR genes. Application of MeJA resulted in the expression of JAmyb in cv. Nakdong being induced faster than in untreated plants in response to M. grisea infection. In contrast, the expression pattern of the PR and JAmyb genes in response to C. miyabeanus was nearly identical between cvs. Nakdong and Tetep, and neither BTH nor MeJA treatment significantly modified their expression patterns in response to C. miyabeanus infection. Our results suggest that rice employs distinct mechanisms for its defense against M. grisea versus C. miyabeanus.

Calcium restores prepenetration morphogenesis abolished by polyamines in Colletotrichum gloeosporioides infecting red pepper.

Colletotrichum gloeosporioides forms a specialized infection structure, an appressorium, to infect its host, red pepper. Polyamines (putrescine, spermidine, and spermine) as well as S-adenosyl methionine inhibitor, methylglyoxal-bis-guanyl hydrazone (MGBG), impaired conidial germination and appressorium formation of C. gloeosporioides. Curtailment of cell differentiation by polyamines and MGBG was more evident in conidial germination than in appressorium development. Exogenous addition of calcium restored conidial germination and appressorium formation and expression of calmodulin-encoding gene (CgCaM) inhibited by polyamines. Taken together, proper regulation of intracellular polyamine concentration is indispensable for conidial germination and appressorium formation, and involved in Ca(2+)/calmodulin-dependent signaling pathways of C. gloeosporioides infecting red pepper.

Rhizobacteria-induced resistance perturbs viral disease progress and triggers defense-related gene expression.

Selected strain of nonpathogenic rhizobacterium EXTN-1 from the Bacillus amyloliquefaciens is capable of eliciting broad-spectrum induced systemic resistance (ISR) in several crops that is phenotypically similar to pathogen-induced systemic acquired resistance (SAR). In tobacco (Nicotiana tabacum cv. Samsun-nn), EXTN-1 treatment also perturbs the disease progress by Pepper mild mottle virus (PMMoV), a member of Tobamovirus group. To investigate the defense mechanisms induced by this rhizobacterium, expression patterns of defense-related genes were analyzed. The EXTN-1-treated tobacco plants showed augmented, rapid transcript accumulation of defense-related genes including PR-1a, phenylalanine ammonia-lyase (PAL), and 3-hydroxy-3-methylglutaryl CoA reductase (HMGR) following inoculation with PMMoV. This was the typical phenomenon of potentiation. Accelerated expression of all these genes was subsequently detected in the noninoculated, upper leaves; thus, their expression is associated with the development of both local and systemic resistance. Coordinated reduction of viral genome accumulation was clearly detected in the leaves of tobacco pretreated with EXTN-1. EXTN-1 treatment on Arabidopsis wild type Col-0 resulted in the activation of PR-1 and PDF1.2 at the same time. All these results may indicated that EXTN-1 induces systemic resistance via salicylic acid- and jasmonic acid-dependent pathways and timely recognition followed by rapid counter attack against the viral invasion is the key differences between incompatible interaction and compatible one.

Molecular characterization of a pathogenesis-related protein 8 gene encoding a class III chitinase in rice.

A cDNA encoding a class III chitinase (Oschib1) was isolated from a cDNA library constructed from rice leaves infected with the blast fungus Magnaporthe grisea. The cDNA contains an open reading frame of 861 nucleotides encoding 286 amino acid residues with a pI of 5.06. The deduced amino acid sequence of Oschibl has a high level of similarity with class IIIb chitinases of Gladiolus gandavensis (46%) and Tulipa bakeri (49%). A high level of Oschibl mRNA was detected after inoculation with M. grisea or Xanthomonas oryzae pv. oryzae. Expression of Oschib1 was induced more rapidly when an avirulent strain of M. grisea was inoculated (incompatible interaction) than when a virulent strain was used (compatible interaction). Expression of Oschibl was also induced by treatment of signaling molecules such as salicylic acid, ethylene, and methyl jasmonic acid, and by treatment with H2O2 or CuSO4. The induction patterns of Oschibl expression suggest that Oschib1 may be involved in defense response against pathogen infections and may be classified as a member of pathogenesis-related protein 8 in rice.

Calcium/Calmodulin-Dependent Signaling for Prepenetration Development in Colletotrichum gloeosporioides.

ABSTRACT Colletotrichum gloeosporioides forms a specialized infection structure, an appressorium, for host infection. Contacting hard surface induces appressorium formation in C. gloeosporioides, whereas hydrophobicity of the contact surface does not affect this infection-related differentiation. To determine if the calcium/calmodulin-dependent signaling system is involved in prepenetration morphogenesis in C. gloeosporioides pathogenic on red pepper, effects of calcium chelator (EGTA), phospholipase C inhibitor (neomycin), intracellular calcium modulators (TMB-8 and methoxy verampamil), and calmodulin antagonists (chloroproma-zine, phenoxy benzamine, and W-7) were tested on conidial germination and appressorium formation. Exogenous addition of Ca(2+), regardless of concentration, augmented conidial germination, while appressorial differentiation decreased at higher concentrations. Inhibition of appressorium formation by EGTA was partly restored by the addition of calcium ionophore A23187 or CaCl(2). Calcium channel blockers and calmodulin antagonists specifically reduced appressorium formation at micromolar levels. These results suggest that biochemical processes controlled by the calcium/calmodulin signaling system are involved in the induction of prepenetration morphogenesis in C. gloeosporioides pathogenic on red pepper.

Transcriptome analysis of grain-filling caryopses reveals the potential formation mechanism of the rice sugary mutant.

A sugary mutant with low total starch and high sugar contents was compared with its wild type Sindongjin for grain-filling caryopses. In the present study, developing seeds of Sindongjin and sugary mutant from the 11th day after flowering (DAF) were subjected to RNA sequencing (RNA-Seq). A total of 30,385 and 32,243 genes were identified in Sindongjin and sugary mutant. Transcriptomic change analysis showed that 7713 differentially expressed genes (DEGs) (log2 fold change ≥1, false discovery rate (FDR)≤0.001) were identified based on our RNA-Seq data, with 7239 genes up-regulated and 474 down-regulated in the sugary mutant. A large number of DEGs were found related to metabolic, biosynthesis of secondary metabolites, plant-pathogen interaction, plant hormone signal transduction and starch/sugar metabolism. Detailed pathway dissection and quantitative real time PCR (qRT-PCR) demonstrated that most genes involved in sucrose to starch synthesis are up-regulated, whereas the expression of the ADP-glucose pyrophosphorylase small subunit (OsAGPS2b) catalyzing the first committed step of starch biosynthesis was specifically inhibited during the grain-filling stage in sugary mutant. Further analysis suggested that the OsAGPS2b is a considerable candidate gene responsible for phenotype of sugary mutant.