MoDHX35, a DEAH-Box Protein, Is Required for Appressoria Formation and Full Virulence of the Rice Blast Fungus, Magnaporthe oryzae.

The DExD/H-box protein family encompasses a large number of RNA helicases that are involved in RNA metabolism and a variety of physiological functions in different species. However, there is limited knowledge of whether DExD/H-box proteins play a role in the pathogenicity of plant fungal pathogens. In the present work, the DExD/H-box protein MoDHX35, which belongs to the DEAH subfamily, was shown to be crucial in appressoria formation and full virulence of the rice blast fungus, Magnaporthe oryzae. The predicted protein sequence of MoDHX35 had typical DEAH-box domains, showed 47% identity to DHX35 in Homo species, but had no orthologs in Saccharomyces cerevisiae. Deletion of the MoDHX35 gene resulted in reduced tolerance of the mutants to doxorubicin, a nucleic acid synthesis disturbing agent, suggesting the involvement of MoDHX35 in RNA metabolism. MoDHX35-deleted mutants exhibited normal vegetative growth, conidia generation and conidial germination, but showed a reduced appressorium formation rate and attenuated virulence. Our work demonstrates the involvement of DEAH-box protein functions in the pathogenicity of plant fungal pathogens.

Adenylsuccinate Synthetase MoADE12 Plays Important Roles in the Development and Pathogenicity of the Rice Blast Fungus.

Purines are basic components of nucleotides in living organisms. In this study, we identified the ortholog of adenylosuccinate synthase MoADE12 in Magnaporthe oryzae by screening for growth-defective T-DNA insertional mutants. Gene replacement was performed to investigate the biological role of MoADE12. Δmoade12 mutants were adenine auxotrophs that failed to produce conidia, and showed reduced perithecia formation and pathogenicity. Moreover, the Δmoade12 mutant was hypersensitive to Congo red and oxidants, indicating that MoADE12 was required for cell wall integrity and oxidative stress resistance. Transcriptomic analysis identified the underlying mechanisms and indicated that several pathogenicity-related genes were regulated in the Δmoade12 mutant. Therefore, our data suggest that the adenylosuccinate synthase MoADE12 is involved in the de novo AMP biosynthesis pathway and is important for conidiation and pathogenicity in the rice blast fungus.

The flavohemoglobin gene MoFHB1 is involved in the endurance against nitrosative stress in Magnaporthe oryzae.

Nitric oxide (NO) homeostasis plays a versatile role in pathogen-host interactions. To maintain NO homeostasis in favor of pathogens, microbes have evolved NO degradation systems besides NO synthesis pathway, in which the flavohemoglobin and S-nitrosoglutathione (GSNO) reductase are two key enzymes. We previously proved that MoSFA1, a GSNO reductase, is required for the growth and pathogenicity in Magnaporthe oryzae. In the present work, MoFHB1, a flavohemoglobin-encoding gene in M. oryzae was functionally characterized. Although the expression of the MoFHB1 gene was developmentally regulated during conidial germination and appressorium development, disruption of MoFHB1 did not change vegetative growth, conidiation and virulence. However, compared with the Δmosfa1 mutant, the Δmofhb1 mutant was significantly more sensitive to NO stress, and the expression of MoSFA1 gene in the Δmofhb1 mutant was significantly upregulated. Double deletion of MoSFA1 and MoFHB1 led to greater sensitivity of the fungus to NO stress than either of the single gene mutant, but no further reduction in pathogenicity was found compared with that of Δmosfa1 mutant. Taken together, MoFHB1 played an important role in NO detoxification but was dispensable for virulence of M. oryzae.

Monitoring peroxisome dynamics using enhanced green fluorescent protein labeling in Alternaria alternata.

Brown leaf spot on tobacco is a serious fungal disease caused by Alternaria alternata. Peroxisomes are organelles playing an important role in the development and infection of plant pathogenic fungi. But, until now, there is no report on the peroxisome dynamics during the conidia germination of A. alternata. To evaluate the roles of peroxisome in the development of the fungus, in the present work, an enhanced green fluorescent protein (eGFP) cassette tagged with peroxisome targeting signal 2 (PTS2) was integrated into A. alternata to label the organelles, and an eGFP cassette carrying a nuclear located signal (NLS) was performed parallelly. The transformants containing the fusions emitted fluorescence in punctate patterns. The fluorescence of eGFP-PTS2 was distributed exactly in the peroxisomes while those of eGFP-NLS were located in the nucleus. Typical AaGB transformants were selected to be investigated for the peroxisome dynamics. The results showed that during spore germination, the number of peroxisomes in the spores decreased gradually, but increased in the germ tubes. In addition, when the transformants were cultured on lipid media, the numbers of peroxisomes increased significantly, and in a larger portion, present in striped shapes. These findings give some clues for understanding the peroxisomal functions in the development of A. alternata.

The peroxins BcPex8, BcPex10, and BcPex12 are required for the development and pathogenicity of Botrytis cinerea.

Peroxisomes have been proved playing roles in infection of several plant pathogens. Although the contribution of a portion of peroxins in pathogenicity was demonstrated, most of them are undocumented in fungi, especially, Botrytis cinerea. The homologs of Pex8, Pex10, and Pex12 in B. cinerea were functionally characterized in this work using gene disruption strategies. Compared with the wild-type strain (WT), the Δbcpex8, Δbcpex10, and Δbcpex12 mutants exhibited significant reduction in melanin production, fatty acid utilization, and decreased tolerance to high osmotic pressure and reactive oxygen species (ROS). The mycelial growth and conidiation of were significantly inhibited in Δbcpex8, Δbcpex10, and Δbcpex12 strains. The mycelial growth rates of Δbcpex8, Δbcpex10, and Δbcpex12 were reduced by 32, 35, and 34%, respectively, compared with WT and ectopic transformant (ET), and the conidiation was reduced by approximately 89, 27, and 88%, respectively. The conidial germination, germ tube elongation, and the formation of initiate infection structures (IFSs) were also reduced by the deletion of the genes. The pathogenicity was tested on the leaves of tobacco and strawberry, and fruits of tomato. On the leaves of tobacco and strawberry, the Δbcpex8, Δbcpex10, and Δbcpex12 mutants could not induce necrotic lesions, and the lesions on tomato fruits infected with the mutants were significantly reduced than those of the wide type. The results indicated that BcPEX8, BcPEX10, and BcPEX12 are indispensable for the development and pathogenicity of B. cinerea.

MAT Loci Play Crucial Roles in Sexual Development but Are Dispensable for Asexual Reproduction and Pathogenicity in Rice Blast Fungus Magnaporthe oryzae.

Magnaporthe oryzae, a fungal pathogen that causes rice blast, which is the most destructive disease of rice worldwide, has the potential to perform both asexual and sexual reproduction. MAT loci, consisting of MAT genes, were deemed to determine the mating types of M. oryzae strains. However, investigation was rarely performed on the development and molecular mechanisms of the sexual reproduction of the fungus. In the present work, we analyzed the roles of two MAT loci and five individual MAT genes in the sex determination, sexual development and pathogenicity of M. oryzae. Both of the MAT1-1 and MAT1-2 loci are required for sex determination and the development of sexual structures. MAT1-1-1, MAT1-1-3 and MAT1-2-1 genes are crucial for the formation of perithecium. MAT1-1-2 impacts the generation of asci and ascospores, while MAT1-2-2 is dispensable for sexual development. A GFP fusion experiment indicated that the protein of MAT1-1-3 is distributed in the nucleus. However, all of the MAT loci or MAT genes are dispensable for vegetative growth, asexual reproduction, pathogenicity and pathogenicity-related developments of the fungus, suggesting that sexual reproduction is regulated relatively independently in the development of the fungus. The data and methods of this work may be helpful to further understand the life cycle and the variation of the fungus.

Succinyl-proteome profiling of Pyricularia oryzae, a devastating phytopathogenic fungus that causes rice blast disease.

Pyricularia oryzae is the pathogen for rice blast disease, which is a devastating threat to rice production worldwide. Lysine succinylation, a newly identified post-translational modification, is associated with various cellular processes. Here, liquid chromatography tandem-mass spectrometry combined with a high-efficiency succinyl-lysine antibody was used to identify the succinylated peptides in P. oryzae. In total, 2109 lysine succinylation sites in 714 proteins were identified. Ten conserved succinylation sequence patterns were identified, among which, K*******Ksuc, and K**Ksuc, were two most preferred ones. The frequency of lysine succinylation sites, however, greatly varied among organisms, including plants, animals, and microbes. Interestingly, the numbers of succinylation site in each protein of P. oryzae were significantly greater than that of most previous published organisms. Gene ontology and KEGG analysis showed that these succinylated peptides are associated with a wide range of cellular functions, from metabolic processes to stimuli responses. Further analyses determined that lysine succinylation occurs on several key enzymes of the tricarboxylic acid cycle and glycolysis pathway, indicating that succinylation may play important roles in the regulation of basal metabolism in P. oryzae. Furthermore, more than 40 pathogenicity-related proteins were identified as succinylated proteins, suggesting an involvement of succinylation in pathogenicity. Our results provide the first comprehensive view of the P. oryzae succinylome and may aid to find potential pathogenicity-related proteins to control the rice blast disease. Significance Plant pathogens represent a great threat to world food security, and enormous reduction in the global yield of rice was caused by P. oryzae infection. Here, the succinylated proteins in P. oryzae were identified. Furthermore, comparison of succinylation sites among various species, indicating that different degrees of succinylation may be involved in the regulation of basal metabolism. This data facilitates our understanding of the metabolic pathways and proteins that are associated with pathogenicity.

Genomic analysis of hybrid rice varieties reveals numerous superior alleles that contribute to heterosis.

Exploitation of heterosis is one of the most important applications of genetics in agriculture. However, the genetic mechanisms of heterosis are only partly understood, and a global view of heterosis from a representative number of hybrid combinations is lacking. Here we develop an integrated genomic approach to construct a genome map for 1,495 elite hybrid rice varieties and their inbred parental lines. We investigate 38 agronomic traits and identify 130 associated loci. In-depth analyses of the effects of heterozygous genotypes reveal that there are only a few loci with strong overdominance effects in hybrids, but a strong correlation is observed between the yield and the number of superior alleles. While most parental inbred lines have only a small number of superior alleles, high-yielding hybrid varieties have several. We conclude that the accumulation of numerous rare superior alleles with positive dominance is an important contributor to the heterotic phenomena.

Expression patterns of defense genes in resistance of the panicles exserted from the caulis and from the tillers to neck blast in rice.

The rice variety Xiushui227 is resistant to neck blast in the panicles exserted from the caulis but susceptible in the panicles from the tillers, however, the other variety Xiushui09 is susceptible to neck blast in the panicles from the caulis but resistant in the panicles from the tillers. These two varieties were used to analyze the expression patterns of defense genes in the panicles from the caulis and the first first-class tiller at the preliminary heading stage, after inoculating the necks in vitro with Magnaporthe oryzae, respectively. All defense genes (pathogenesis-related genes PR1a, Gns1 (1,3; 1,4-ß-glucanase), Cht-1 (chitinase), PR4, PR5, and PR10a, secondary metabolite pathway genes PAL (phenylalanine ammonia-lyase), CHS (chalcone synthase), and LOX (lipoxygenase), and oxidative stress-related protein genes POX22.3 (peroxidase), and PPO (polyphenol oxidase)) used in this experiment except Cht-1 and PR5 could participate in defending Xiushui227 against neck blast in the panicles from the caulis. All defense genes used in this study except Cht-1, PR10a, and PPO may play roles in defending Xiushui09 against neck blast in the panicles from the tillers.

Expression of defense genes and antioxidant defense responses in rice resistance to neck blast at the preliminary heading stage and full heading stage.

The rice variety Xiushui227 is resistant to neck blast at three crucial panicle stages (the booting stage, the preliminary heading stage (PHS), and the full heading stage (FHS)) that controlling neck blast. The other rice variety Liangyou6326 is susceptible to neck blast at these three panicle stages. These two varieties were used to analyze the expression patterns of defense genes and antioxidant defense responses at the PHS and FHS, after inoculating the necks in vitro with Magnaporthe oryzae, respectively. All defense genes (pathogenesis-related genes PR1a, Gns1 (1,3; 1,4-ß-glucanase), Cht-1 (chitinase), PR4, PR5, and PR10a, secondary metabolite pathway genes PAL (phenylalanine ammonia-lyase), CHS (chalcone synthase), and LOX (lipoxygenase), and oxidative stress-related protein genes POX22.3 (peroxidase), and PPO (polyphenol oxidase)) used in this experiment except Cht-1, CHS and PPO could mainly play important roles in the resistance to neck blast at the PHS in Xiushui227, and CHS and PPO may primarily participate in fighting back against to neck blast at the FHS. Conversely, PR1a, Cht-1, PR4, PR10a, CHS, LOX-RLL, and PPO could chiefly play participate in defending Liangyou6326 against neck blast at the FHS, and PR5, PAL, and POX22.3 may be primarily involved in fighting back against to neck blast at the PHS. Furthermore, the antioxidant enzymes superoxide dismutase, peroxidase, and catalase may mainly participate in defending Xiushui227 against neck blast at the PHS and defending Liangyou6326 against neck blast at the FHS, respectively. Therefore oxidative damage is less at the PHS in Xiushui227 and at the FHS in Liangyou6326, respectively.

Expression of defense genes and activities of antioxidant enzymes in rice resistance to rice stripe virus and small brown planthopper.

The rice variety Tai06-1 is resistant to rice stripe disease and Xiushui63 is a highly susceptible rice variety to this disease. These two varieties were used to analyze the expression patterns of defense genes and antioxidant defense responses at the seedling stage, upon feeding with viruliferous small brown planthopper (SBPH) and nonviruliferous SBPH, respectively. The expression levels of CP (coat protein) gene of rice stripe virus (RSV) were higher upon feeding with viruliferous SBPH in Xiushui63 than in Tai06-1 throughout most of the experimental period, suggesting that RSV replicaiton is disturbed in Tai06-1 but not in Xiushui63, therefore, the resistance to RSV is higher in Tai06-1 than in Xiuhsui63. We found that defense genes PR1a (pathogenesis-related class 1a), PAL (phenylalanine ammonia-lyase), and CHS (chalcone synthase) may play roles in the defense responses to both RSV and SBPH in Tai06-1, and PR4 and PR10a may only participate in defending against SBPH attack but not against RSV infection in Tai06-1. Our data reveal that Gns1 (1,3; 1,4-ß-glucanase) may participate in the defense responses to both RSV and SBPH in Xiushui63 but not in Tai06-1, and LOX (lipoxygenase) may only participate in defending against to SBPH in both Tai06-1 and Xiushui63. The antioxidant enzymes superoxide dismutase, peroxidase, catalase, hydrogen peroxide, and malondialdehyde coordinately participate in the resistance to RSV in Tai06-1, and that oxidative damage is less in Tai06-1 than in Xiushui63.

Overexpression of rice WRKY89 enhances ultraviolet B tolerance and disease resistance in rice plants.

WRKY proteins are a large family of transcriptional regulators involved in a variety of biological processes in plants. Here we report functional characterization of a rice WRKY gene, OsWRKY89. RNA gel blot analysis indicated that OsWRKY89 was strongly induced by treatments of methyl jasmonate and UV-B radiation. The transient expression analysis of the OsWRKY89-eGFP reporter in onion epidermal cells revealed that OsWRKY89 was targeted to nuclei. Transcriptional activity assays of OsWRKY89 and its mutants fused with a GAL4 DNA binding domain indicated that the 67 C-terminal amino acids were required for the transcriptional activation and that the leucine zipper region at the N-terminus enhanced its transcriptional activity. Overexpression of OsWRKY89 led to growth retardation at the early stage and reduction of internode length. Scanning electron microscopy revealed an increase in wax deposition on leaf surfaces of the OsWRKY89 overexpression lines and a decrease in wax loading in the RNAi-mediated OsWRKY89 suppression lines. Moreover, extractable and cell-wall-bound phenolic compounds were decreased in the overexpressor lines, but its SA levels were increased. Lignin staining showed an increase in lignification in culms of the overexpressor lines. Interestingly, overexpression of the OsWRKY89 gene enhanced resistance to the rice blast fungus and white-backed planthopper as well as tolerance to UV-B irradiation. These results suggest that OsWRKY89 plays an important role in response to biotic and abiotic stresses.