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OsWRKY76 encodes a group IIa WRKY transcription factor of rice. The expression of OsWRKY76 was induced within 48h after inoculation with rice blast fungus (Magnaporthe oryzae), and by wounding, low temperature, benzothiadiazole, and abscisic acid. Green fluorescent protein-fused OsWRKY76 localized to the nuclei in rice epidermal cells. OsWRKY76 showed sequence-specific DNA binding to the W-box element in vitro and exhibited W-box-mediated transcriptional repressor activity in cultured rice cells. Overexpression of OsWRKY76 in rice plants resulted in drastically increased susceptibility to M. oryzae, but improved tolerance to cold stress. Microarray analysis revealed that overexpression of OsWRKY76 suppresses the induction of a specific set of PR genes and of genes involved in phytoalexin synthesis after inoculation with blast fungus, consistent with the observation that the levels of phytoalexins in the transgenic rice plants remained significantly lower than those in non-transformed control plants. Furthermore, overexpression of OsWRKY76 led to the increased expression of abiotic stress-associated genes such as peroxidase and lipid metabolism genes. These results strongly suggest that OsWRKY76 plays dual and opposing roles in blast disease resistance and cold tolerance.

Abstract Rice blast fungus (Pyricularia oryzae) is a heterothallic ascomycete that causes the most destructive disease in cultivated rice worldwide. This fungus reproduces sexually and asexually, and its mating type is determined by the MAT1 locus, MAT1-1 or MAT1-2. Interestingly, most rice-infecting field isolates show a loss of female fertility, but the MAT1 locus is highly conserved in female-sterile isolates. In this study, we performed a functional analysis of MAT1 using the CRISPR/Cas9 system in female- and male-fertile isolates and female-sterile (male-fertile) isolates. Consistent with a previous report, MAT1 was essential for sexual reproduction but not for asexual reproduction. Meanwhile, deletion mutants of MAT1-1-1, MAT1-1-2, and MAT1-1-3 exhibited phenotypes different from those of other previously described isolates, suggesting that the function of MAT1-1 genes and/or their target genes in sexual reproduction differs among strains or isolates. The MAT1 genes, excluding MAT1-2-6, retained their functions even in female-sterile isolates, and deletion mutants lead to loss or reduction of male fertility. Although MAT1 deletion did not affect microconidia (spermatia) production, microconidia derived from the mutants could not induce perithecia formation. These results indicated that MAT1 is required for microconidia-mediated male fertility in addition to female fertility in P. oryzae . MAT1 is required for microconidia-mediated male fertility in addition to female fertility in the rice blast fungus Pyricularia oryzae.

Plant pathogens evade basal defense systems and attack different organs and tissues of plants. Genetic engineering of plants with genes that confer resistance against pathogens is very effective in pathogen control. Conventional breeding for disease resistance in ornamental crops is difficult and lagging relative to that in non-ornamental crops due to an inadequate number of disease-resistant genes. Therefore, genetic engineering of these plants with defense-conferring genes is a practical approach. We used rice BSR2 encoding CYP78A15 for developing transgenic Torenia fournieri Lind. lines. The overexpression of BSR2 conferred resistance against two devastating fungal pathogens, Rhizoctonia solani and Botrytis cinerea. In addition, BSR2 overexpression resulted in enlarged flowers with enlarged floral organs. Histological observation of the petal cells suggested that the enlargement in the floral organs could be due to the elongation and expansion of the cells. Therefore, the overexpression of BSR2 confers broad-spectrum disease resistance and induces the production of enlarged flowers simultaneously. Therefore, this could be an effective strategy for developing ornamental crops that are disease-resistant and economically more valuable.

Candidatus Liberibacter solanacearum (Lso) is a plant pathogenic bacterium and the causal agent of potato ‘zebra chip’ and carrot Lso diseases. Because this pathogen is suspected to be transmitted via carrot seeds, seed Lso disinfection is important for seed transportation and distribution worldwide. However, since Lso is unculturable, disinfection efficacy cannot be confirmed using bacterial culture assays. Here, we focused on Lso RNA to detect viable Lso cells in Lso-contaminated carrot seeds. Because bacterial RNA is only transcribed in living bacteria, we reasoned that non-detection of Lso RNA after seed disinfection would signify that Lso had not survived, which could be confirmed using reverse-transcription quantitative PCR. Indeed, when six culturable seed-transmitted bacterial strains were all killed by autoclaving or sodium hypochlorite treatment, neither RNA nor colony formation was detected, confirming that non-detection of RNA indicated successful sterilization. When Lso-contaminated carrot seeds were separately treated with five disinfection methods, four, including hot water treatment, resulted in non-detection of Lso RNA, even though carrot RNA was detected and the seeds germinated normally. We suggest that an RNA-based PCR assay is suitable for identifying the presence of plant pathogenic bacteria, which will be useful for screening disinfection methods for Lso-contaminated seeds.

A histological study of red stripe of rice was conducted to elucidate the mode of infection of the causal bacterium Microbacterium sp. When pin-point-sized spots first appeared at 3 days after inoculation, the bacterial cells had entered through stomata and multiplied in the intercellular spaces of substomatal parenchymatous tissues. With the early appearance of small yellow spots at 4 to 5 days after inoculation, the bacterium was detected in some xylem vessels as well as in parenchymatous tissues, and it had apparently translocated directly from parenchymatous tissues to transverse vascular systems through spiral vessel walls. With the appearance of typical red stripe symptoms comprised of orange lesions and halos at 8 days after inoculation, bacterial masses were present in transverse and longitudinal vascular bundles in areas with orange lesions. In the areas with orange to light brown spots, granules that stained dark blue using Stoughton's method appeared in the protoplasm of the host parenchymatous cells, which later became necrotic. In halo areas, bacterial masses were observed only in some cases, and chloroplasts were disorganized. Bacterial infection was also confirmed by observing sections of naturally infected samples, and the distribution of bacteria was much more extensive than in artificially inoculated samples.

Bacterial fruit blotch of cucurbits is a destructive disease caused by Acidovorax avenae subsp. citrulli, which is a typical seedborne pathogen. In seed health testing for this disease, we have detected many strains of Acidovorax with some differences from A. avenae subsp. citrulli. Their 16S rRNA sequences were divided into six types. The most common sequence was completely consistent with that of A. avenae subsp. avenae originally isolated from rice. The other sequences were over 99% similar but not identical to those of A. avenae subsp. avenae and A. avenae subsp. citrulli. Some commercialized antibodies against A. avenae subsp. citrulli reacted with several of these strains. Some of these strains incited yellow spots or brownish water-soaked lesions mainly on young true leaves of cucumber and squash after spray inoculation. Histological observations showed that these strains entered the leaf tissues of cucurbit plants through stomata and multiplied in the intercellular spaces of parenchymatous tissues as well as in the vascular tissues. The amount of bacterial multiplication and spread in the tissues differed among the strains, presumably reflecting their ability to induce symptoms. These isolated strains are therefore different from A. avenae subsp. citrulli, and their potential threat to the cultivation of cucurbits is lower than that of A. avenae subsp. citrulli.

Xanthomonas oryzae pv. oryzae, the causal agent of bacterial leaf blight of rice, was subjected to transposon mutagenesis to generate mutants defective in pathogenicity. A novel mutant 74M913 was attenuated in virulence but retained its ability to cause the hypersensitive response in leaf blight-resistant rice and tomato. Cloning and sequence analysis revealed that the transposon in 74M913 was inserted in a gene homologous to the phosphoglucose isomerase (pgi) gene of X. axonopodis pv. citri. Growth of the mutant in a synthetic medium containing fructose or xylose as a sole carbohydrate source was much reduced, indicating the transposon disrupted pgi function. The interaction between expression of pgi and hypersensitive response and pathogenicity (hrp) genes was investigated because we had demonstrated previously that expression of hrp genes of X. oryzae pv. oryzae is induced in a synthetic medium containing xylose. However, pgi and the hrp gene (hrcU) were expressed independently. This study suggests that PGI is involved in pathogenicity of X. oryzae pv. oryzae.

A wilt disease of the model legume Lotus japonicus was observed in a greenhouse in Tokyo, Japan in May 2004. Roots of diseased plants were rotted and dark brown with lesions spreading to lower stems and leaves, resulting in rapid plant death. The causal agent was identified as Fusarium solani based on the morphology. Sequence analysis of rDNA supported the identification. Inoculation of roots of healthy plants with conidia reproduced characteristic disease symptoms, and F. solani was reisolated from lesions, satisfying Koch's postulates. The isolate also caused chlorotic to necrotic lesions on leaves of healthy plants after wound-inoculation. Infection by F. solani of leaves of L. japonicus was confirmed histologically. Mycelia were observed in the intercellular spaces of parenchymatous tissues in the lesion area and the surrounding tissues. This is the first report of fungal disease on L. japonicus satisfying Koch's postulates. We named it “Fusarium root rot of L. japonicus” as a new disease. The compatibility of L. japonicus and F. solani is expected to form a novel pathosystem for studying interactions between legumes and fungal pathogens.

The clustered hrp genes encoding the type III secretion system in the Japanese strains MAFF301237 and MAFF311018 of Xanthomonas oryzae pv. oryzae were sequenced and compared. The strains differ in their pathogenicity, location, and year of isolation. A 30-kbp sequence comprising 29 open reading frames (ORFs) was identical in its structural arrangement in both strains but differed from X. campestris pv. campestris, X. axonopodis pv. citri, and X. axonopodis pv. glycines in certain genes located between the hpaB-hrpF interspace region. The DNA sequence and the putative amino acid sequence in each ORF was also identical in both X. oryzae pv. oryzae strains as were the PIP boxes and the relative sequences. These facts clearly showed that the structure of the hrp gene cluster in X. oryzae pv. oryzae is unique. [PUBLICATION ABSTRACT]