Comparative Analysis of the Genomes of Two Field Isolates of the Rice Blast Fungus Magnaporthe oryzae

Rice blast caused by Magnaporthe oryzae is one of the most destructive diseases of rice worldwide. The fungal pathogen is notorious for its ability to overcome host resistance. To better understand its genetic variation in nature, we sequenced the genomes of two field isolates, Y34 and P131. In comparison with the previously sequenced laboratory strain 70-15, both field isolates had a similar genome size but slightly more genes. Sequences from the field isolates were used to improve genome assembly and gene prediction of 70-15. Although the overall genome structure is similar, a number of gene families that are likely involved in plant-fungal interactions are expanded in the field isolates. Genome-wide analysis on asynonymous to synonymous nucleotide substitution rates revealed that many infection-related genes underwent diversifying selection. The field isolates also have hundreds of isolate-specific genes and a number of isolate-specific gene duplication events. Functional characterization of randomly selected isolate-specific genes revealed that they play diverse roles, some of which affect virulence. Furthermore, each genome contains thousands of loci of transposon-like elements, but less than 30% of them are conserved among different isolates, suggesting active transposition events in M. oryzae. A total of approximately 200 genes were disrupted in these three strains by transposable elements. Interestingly, transposon-like elements tend to be associated with isolate-specific or duplicated sequences. Overall, our results indicate that gain or loss of unique genes, DNA duplication, gene family expansion, and frequent translocation of transposon-like elements are important factors in genome variation of the rice blast fungus.     

Inhibition of resistance gene-mediated defense in rice by Xanthomonas oryzae pv. oryzicola

Xanthomonas oryzae pv. oryzae and the closely related X. oryzae pv. oryzicola cause bacterial blight and bacterial leaf streak of rice, respectively. Although many rice resistance (R) genes and some corresponding avirulence (avr) genes have been characterized for bacterial blight, no endogenous avr/R gene interactions have been identified for leaf streak. Genes avrXa7 and avrXa10 from X. oryzae pv. oryzae failed to elicit the plant defense-associated hypersensitive reaction (HR) and failed to prevent development of leaf streak in rice cultivars with the corresponding R genes after introduction into X. oryzae pv. oryzicola despite the ability of this pathovar to deliver an AvrXa10:Cya fusion protein into rice cells. Furthermore, coinoculation of X. oryzae pv. oryzicola inhibited the HR of rice cultivar IRBB10 to X. oryzae pv. oryzae carrying avrXa10. Inhibition was quantitative and dependent on the type III secretion system of X. oryzae pv. oryzicola. The results suggest that one or more X. oryzae pv. oryzicola type III effectors interfere with avr/R gene-mediated recognition or signaling and subsequent defense response in the host. Inhibition of R gene-mediated defense by X. oryzae pv. oryzicola may explain, in part, the apparent lack of major gene resistance to leaf streak.     

Plasmid and pathogenicity in Xanthomonas oryzae pathovar oryzae, the bacterial blight pathogen of Oryza sativa

Xanthomonas oryzae pv. oryzae , the causative agent for bacterial leaf blight of rice, comprises diverse groups of strains differing in biochemical and pathological characteristics. A collection of X.o . pv. oryzae strains differing in geographical origin was screened for the presence of plasmids. Out of 17 isolates of X.o. pv. oryzae , 14 harboured plasmids of which two isolates (XoP5, XoC26) had two plasmids each and one isolate (XoR20) had three. The remaining isolates contained a single plasmid of identical mobility. Finger print analysis of plasmids was carried out using Eco RI for 10 isolates. The restriction fragment pattern was distinct for each isolate. They were classified under three groups based on cluster analysis using the unweighted pair group method with averages (UPGMA). Of the 18 plasmids, the plasmid pMA36 ( X.o. pv. oryzae XoC36) was further characterized. This plasmid was cured by acridine orange at the frequency rate of 10%. The cured strain was transformed with pMA36 at a frequency of 2.3 times 10² transformants μg^-1 of plasmid DNA. The plasmid-cured strain was virulent on rice but symptom development was delayed when compared to wild and transformed strains. The wild type strain ( X.o. pv. oryzae XoC36) was resistant to ampicillin, carbenicillin and rifampicin whereas the cured strain was resistant to carbenicillin and rifampicin but sensitive to ampicillin. The transformant was resistant to the three antibiotics indicating that the plasmid pMA36 codes for ampicillin resistance. The plasmid influenced the pathogenicity of X.o. pv. oryzae.     

Diverse members of the AvrBs3/PthA family of type III effectors are major virulence determinants in bacterial blight disease of rice

AvrXa7 is a member of the avBs3/pthA gene family and the only known type III secretion system effector gene from Xanthomonas oryzae pv. oryzae with a major contribution to bacterial growth and lesion formation in bacterial blight disease of rice. We examined the general requirement for effectors of the AvrBs3/PthA family in bacterial blight of rice by identifying effectors from diverse strains of the pathogen. Inactivation of single effector genes in representative strains from Japan, Korea, and the Philippines resulted in severely limited growth in plants. Five strains harbored one gene of the avrBs3/pthA family, while one strain had two genes with the equivalent virulence activity of avrXa7. Sequence analysis revealed three genes with unique repeat arrangements in comparison to avrXa7. Comparison of the repetitive regions revealed a potential motif for the group that was also present in the repetitive region of avrBs3. However, the repetitive region of avrBs3 could not support virulence activity but, in combination with the C-terminal coding region of avrXa7, triggered a Xa7-dependent avirulence reaction. The results revealed diverse members of the avrBs3/pthA gene family with virulence activity in X. oryzae pv. oryzae and supported the hypothesis that bacterial blight disease of rice is highly dependent on a single class of type III effectors. The results also indicated that avrXa7 avirulence specificity is separable from virulence activity.     

A multilocus gene genealogy concordant with host preference indicates segregation of a new species, Magnaporthe oryzae, from M. grisea

Magnaporthe oryzae is described as a new species distinct from M. grisea. Gene trees were inferred for Magnaporthe species using portions of three genes: actin, beta-tubulin, and calmodulin. These gene trees were found to be concordant and distinguished two distinct clades within M. grisea. One clade is associated with the grass genus Digitaria and is therefore nomenclaturally tied to M. grisea. The other clade is associated with Oryza sativa and other cultivated grasses and is described as a new species, M. oryzae. While no morphological characters as yet distinguish them, M. oryzae is distinguished from M. grisea by several base substitutions in each of three loci as well as results from laboratory matings; M.oryzae and M. grisea are not interfertile. Given that M. oryzae is the scientifically correct name for isolates associated with rice blast and grey leaf spot, continued use of M. grisea for such isolates would require formal nomenclatural conservation.     

Characterization of the hrpF pathogenicity peninsula of Xanthomonas oryzae pv. oryzae

The hrp gene cluster of Xanthomonas spp. contains genes for the assembly and function of a type III secretion system (TTSS). The hrpF genes reside in a region between hpaB and the right end of the hrp cluster. The region of the hrpF gene of Xanthomonas oryzae pv. oryzae is bounded by two IS elements and also contains a homolog of hpaF of X. campestris pv. vesicatoria and two newly identified genes, hpa3 and hpa4. A comparison of the hrp gene clusters of different species of Xanthomonas revealed that the hrpF region is a constant yet more variable peninsula of the hrp pathogenicity island. Mutations in hpaF, hpa3, and hpa4 had no effect on virulence, whereas hrpF mutants were severely reduced in virulence on susceptible rice cultivars. The hrpF genes from X. campestris pv. vesicatoria, X. campestris pv. campestris, and X. axonopodis pv. citri each were capable of restoring virulence to the hrpF mutant of X. oryzae pv. oryzae. Correspondingly, none of the Xanthomonas pathovars with hrpF from X. oryzae pv. oryzae elicited a hypersensitive reaction in their respective hosts. Therefore, no evidence was found for hrpF as a host-specialization factor. In contrast to the loss of Bs3-dependent reactions by hrpF mutants of X. campestris pv. vesicatoria, hrpF mutants of X. oryzae pv. oryzae with either avrXa10 or avrXa7 elicited hypersensitive reactions in rice cultivars with the corresponding R genes. A double hrpFxoo-hpa1 mutant also elicited an Xa10-dependent resistance reaction. Thus, loss of hrpF, hpal, or both may reduce delivery or effectiveness of type III effectors. However, the mutations did not completely prevent the delivery of effectors from X. oryzae pv. oryzae into the host cells.     

水稻品种多样性田间稻瘟病菌群体遗传结构分析

利用稻瘟病菌的一段倒位重复序列Pot2设计的一对引物,采用rep—PCR分子指纹技术对来自石屏县净种杂交稻田块、净种糯稻田块以及间种杂交稻糯稻田间的251个稻瘟病单孢分离菌株进行扩增．结果表明,所有供试菌株均分别扩增到9—17条DNA带,大小从400bp到23kb左右,但大多数带主要集中在5—10kb之间．所有菌株共扩增出的DNA指纹带中,约65％的为多态性DNA带,35％的为共同扩增带．将供试菌株扩增带诺进行聚类分析,比较间裁与净载田间病菌群体遗传结构的组成差异结果表明,在不同遗传相似水平,菌株遗传宗群复杂度与栽培方式有一定相关性。间栽田间病菌遗传宗群较净栽田问复杂,为3—5个,且优势宗群群不明显;而在净栽糯稻或净栽杂交稻田间遗传宗群较为简单,只有1—3个,且优势宗群明显．本试验结果证明水稻品种多样性有利于稻瘟病菌稳定化选择。     

Instability of the Magnaporthe oryzae avirulence gene AVR-Pita alters virulence

The avirulence gene AVR-Pita of Magnaporthe oryzae determines the efficacy of the resistance gene Pi-ta in rice. The structures of the AVR-Pita alleles in 39 US isolates of M. oryzae were analyzed using polymerase chain reaction. A series of allele-specific primers were developed from the AVR-Pita gene to examine the presence of AVR-Pita. Orthologous alleles of the AVR-Pita gene were amplified from avirulent isolates. Sequence analysis of five alleles revealed three introns at identical positions in the AVR-Pita gene. All five alleles were predicted to encode metalloprotease proteins highly similar to the AVR-Pita protein. In contrast, the same regions of the AVR-Pita alleles were not amplified in the most virulent isolates, and significant variations of DNA sequence at the AVR-Pita allele were verified by Southern blot analysis. A Pot3 transposon was identified in the DNA region encoding the putative protease motif of the AVR-Pita protein from a field isolate B2 collected from a Pi-ta-containing cultivar Banks. These findings show that transposons can contribute to instability of AVR-Pita and is one molecular mechanism for defeating resistance genes in rice cultivar Banks.     

The rice 14-3-3 gene family and its involvement in responses to biotic and abiotic stress.

14-3-3 proteins function as major regulators of primary metabolism and cellular signal transduction in plants. However, their involvement in plant defense and stress responses is largely unknown. In order to better address functions of the rice 14-3-3/GF14 proteins in defense and abiotic stress responses, we examined the rice GF14 family that comprises eight numbers. The phylogenetic comparison with the Arabidopsis 14-3-3 family revealed that the majority of rice GF14s might have evolved as an independent branch. At least four rice GF14 genes, GF14b, GF14c, GF14e and Gf14f were differentially regulated in the interactions of rice-Magnaporthe grisea and rice-Xanthomonas oryzae pv. oryzae, and the incompatible interactions stronger induced the genes than the compatible interactions. These GF14 genes were also induced by the defense compounds, benzothiadiazole, methyl jasmonate, ethephon and hydrogen peroxide. Similarly, they were differentially regulated by salinity, drought, wounding and abscisic acid. Tissue-specific analysis and expression of GF14-YFP fusions revealed that the four GF14 isoforms were expressed with tissue specificity and accumulated differentially in the cytoplasm and nucleus. Our current study provides fundamental information for the further investigation of the rice GF14 proteins.     

Identification and characterization of filamentous fungi isolated from fermentation starters for Hong Qu glutinous rice wine brewing

Hong Qu glutinous rice wine is one of the most popular traditional rice wines in China. Traditionally, this wine is brewed from glutinous rice with the addition of wine fermentation starters (Hong Qu (also called red yeast rice) and White Qu). The objective of this study was to investigate the variability of filamentous fungi associated with traditional fermentation starters through a traditional culture-dependent method and a molecular identification approach. In this study, forty-three filamentous fungi were separated by traditional culture-dependent means (macro- and microscopic characteristics) from 10 fermentation starters and classified into 16 different species based on morphological examination and the internal transcribed spacer (ITS) sequences analysis. It was observed that the genus Aspergillus had the highest number (14 isolates) of isolates followed by Rhizopus (11 isolates), Monascus (5 isolates) and Penicillium (4 isolates). The species R. oryzae, A. niger, A. flavus and M. purpureus were frequently found in wine starter samples, among which R. oryzae was the most frequent species. The enzyme-producing properties (glucoamylase, α-amylase and protease) of all fungal isolates from different starters were also evaluated. A. flavus, R. oryzae and M. purpureus were found to be better glucoamylase producers. A. flavus, R. oryzae and A.oryzae exhibited higher activity of α-amylase. A. flavus and A. oryzae had higher protease activity. However, some fungal isolates of the same species exhibited a significant variability in the production levels for all determined enzyme activity. This study is the first to identify filamentous fungi associated with the starter of Hong Qu glutinous rice wine using both traditional and molecular methods. The results enrich our knowledge of liquor-related micro-organisms, and can be used to promote the development of the traditional fermentation technology.     

Structure, function, and phylogeny of the mating locus in the Rhizopus oryzae complex

The Rhizopus oryzae species complex is a group of zygomycete fungi that are common, cosmopolitan saprotrophs. Some strains are used beneficially for production of Asian fermented foods but they can also act as opportunistic human pathogens. Although R. oryzae reportedly has a heterothallic (+/-) mating system, most strains have not been observed to undergo sexual reproduction and the genetic structure of its mating locus has not been characterized. Here we report on the mating behavior and genetic structure of the mating locus for 54 isolates of the R. oryzae complex. All 54 strains have a mating locus similar in overall organization to Phycomyces blakesleeanus and Mucor circinelloides (Mucoromycotina, Zygomycota). In all of these fungi, the minus (-) allele features the SexM high mobility group (HMG) gene flanked by an RNA helicase gene and a TP transporter gene (TPT). Within the R. oryzae complex, the plus (+) mating allele includes an inserted region that codes for a BTB/POZ domain gene and the SexP HMG gene. Phylogenetic analyses of multiple genes, including the mating loci (HMG, TPT, RNA helicase), ITS1-5.8S-ITS2 rDNA, RPB2, and LDH genes, identified two distinct groups of strains. These correspond to previously described sibling species R. oryzae sensu stricto and R. delemar. Within each species, discordant gene phylogenies among multiple loci suggest an outcrossing population structure. The hypothesis of random-mating is also supported by a 50:50 ratio of plus and minus mating types in both cryptic species. When crossed with tester strains of the opposite mating type, most isolates of R. delemar failed to produce zygospores, while isolates of R. oryzae produced sterile zygospores. In spite of the reluctance of most strains to mate in vitro, the conserved sex locus structure and evidence for outcrossing suggest that a normal sexual cycle occurs in both species.     

中国大豆疫霉菌遗传多样性的RAPD分析*

采用随机扩增多态性DNA(RAPD)技术,利用13个引物对75个中国大豆疫霉菌分离物和11个美国分离物进行PCR扩增。在78个RAPD标记中,多态性标记为68个,占87.2%。RAPD指纹聚类分析表明,当以相异距离0.3为阈值,86个分离物被划为12个RAPD遗传组,其中J组有54个分离物,占总数的62.8%,包括44个中国分离物和10个美国分离物。在中国大豆疫霉菌群体内,多数分离物之间遗传相似性较低,在DNA水平上存在显著的遗传变异,具有较丰富的遗传多样性。RAPD分组结果未表明大豆疫霉菌DNA多态性特征与病原菌毒力基因构成之间和分离物地理来源之间存在相关性,证明中国不同地区的大豆疫霉菌群体在与大豆品种的互作中发生了广泛的遗传变异,具有DNA遗传进化方向和毒力基因演变的多样性。美国大豆疫霉菌分离物间遗传距离较近,而中国分离物在总体上与美国分离物的遗传距离较远,表明中国大豆疫霉菌具有比较独特的遗传背景。     

Population structure analysis and association mapping of bacterial blight resistance in <Emphasis Type="Italic">indica</Emphasis> rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.) accessions

Bacterial blight, caused by Xanthomonas oryzae pv. oryzae (Xoo), is a serious disease in rice production worldwide. To understand the genetic diversity of bacterial blight resistance a population consisting of 175 indica accessions from nine countries was collected and detected their association between SSR (Simple Sequence Repeat) markers and resistance to six bacterial races. The resistance phenotypes of various rice accessions were evaluated through artificial inoculation under controlled conditions in 2013 and 2014. Association analysis showed that 17 SSR markers were significantly associated with resistance to four bacterial races and the phenotypic variations explained (PVE) ranged from 7.43 to 15.05%. Among the 17 associated SSR markers, two SSR markers located in previously reported genes regions, and 15 SSR markers were newly identified in this study. These results validated a new approach to map resistance genes of rice to bacterial blight. These markers could be used for marker-assisted selection (MAS) in rice bacterial blight resistance breeding programs.     

Adaptive evolution of Xa21 homologs in Gramineae

The XA21 protein has broad spectrum resistance against Xanthomonas oryzae pv. oryzae. Although Xa21-mediated immunity is well characterized, little is known about the origin and evolutionary history of this gene in grasses. Therefore, we analyzed all Xa21 gene homologs in eight whole-genome sequenced rice lines, as well as in four gramineous genomes, rice, Brachypodium, sorghum and maize; using Arabidopsis Xa21 homologs as outgroups, 17, 7, 7 and 3 Xa21 homologs were detected in these four grasses, respectively. Synteny and phylogenetic analysis showed that frequent gene translocation, duplication and/or loss, have occurred at Xa21 homologous loci, suggesting that they have undergone or are undergoing rapid generation of copy number variations. Within the rice species, the high level of nucleotide diversity between Xa21-like orthologs showed a strong association with the presence/absence haplotypes, suggesting that the genetic structure of rice lines plays an important role in the variations between these Xa21-like orthologs. Strongly positive selection was detected in the core region of the leucine-rich repeat domains of the Xa21 subclade among the rice lines, indicating that the rapid gene diversification of Xa21 homologs may be a strategy for a given species to adapt to the changing spectrum of species-specific pathogens.     

中国大豆疫霉菌遗传多样性的RAPD分析

采用随机扩增多态性DNA(RAPD)技术,利用13个引物对75个中国大豆疫霉菌分离物和11个美国分离物进行PCR扩增。在78个RAPD标记中,多态性标记为68个,占87.2%。RAPD指纹聚类分析表明,当以相异距离0.3为阈值,86个分离物被划为12个RAPD遗传组,其中J组有54个分离物,占总数的62.8%,包括44个中国分离物和10个美国分离物。在中国大豆疫霉菌群体内,多数分离物之间遗传相似性较低,在DNA水平上存在显著的遗传变异,具有较丰富的遗传多样性。RAPD分组结果未表明大豆疫霉菌DNA多态性特征与病原菌毒力基因构成之间和分离物地理来源之间存在相关性,证明中国不同地区的大豆疫霉菌群体在与大豆品种的互作中发生了广泛的遗传变异,具有DNA遗传进化方向和毒力基因演变的多样性。美国大豆疫霉菌分离物间遗传距离较近,而中国分离物在总体上与美国分离物的遗传距离较远,表明中国大豆疫霉菌具有比较独特的遗传背景。