Genome organization and evolution of the AVR-Pita avirulence gene family in the Magnaporthe grisea species complex

The avirulence (AVR) gene AVR-Pita in Magnaporthe oryzae prevents the fungus from infecting rice cultivars containing the resistance gene Pi-ta. A survey of isolates of the M. grisea species complex from diverse hosts showed that AVR-Pita is a member of a gene family, which led us to rename it to AVR-Pita1. Avirulence function, distribution, and genomic context of two other members, named AVR-Pita2 and AVR-Pita3, were characterized. AVR-Pita2, but not AVR-Pita3, was functional as an AVR gene corresponding to Pi-ta. The AVR-Pita1 and AVR-Pita2 genes were present in isolates of both M. oryzae and M. grisea, whereas the AVR-Pita3 gene was present only in isolates of M. oryzae. Orthologues of members of the AVR-Pita family could not be found in any fungal species sequenced to date, suggesting that the gene family may be unique to the M. grisea species complex. The genomic context of its members was analyzed in eight strains. The AVR-Pita1 and AVR-Pita2 genes in some isolates appeared to be located near telomeres and flanked by diverse repetitive DNA elements, suggesting that frequent deletion or amplification of these genes within the M. grisea species complex might have resulted from recombination mediated by repetitive DNA elements.     

The Saccharomyces cerevisiae WRN homolog Sgs1p participates in telomere maintenance in cells lacking telomerase

Werner syndrome (WS) is marked by early onset of features resembling aging, and is caused by loss of the RecQ family DNA helicase WRN. Precisely how loss of WRN leads to the phenotypes of WS is unknown. Cultured WS fibroblasts shorten their telomeres at an increased rate per population doubling and the premature senescence this loss induces can be bypassed by telomerase. Here we show that WRN co-localizes with telomeric factors in telomerase-independent immortalized human cells, and further that the budding yeast RecQ family helicase Sgs1p influences telomere metabolism in yeast cells lacking telomerase. Telomerase-deficient sgs1 mutants show increased rates of growth arrest in the G2/M phase of the cell cycle as telomeres shorten. In addition, telomerase-deficient sgs1 mutants have a defect in their ability to generate survivors of senescence that amplify telomeric TG1-3 repeats, and SGS1 functions in parallel with the recombination gene RAD51 to generate survivors. Our findings indicate that Sgs1p and WRN function in telomere maintenance, and suggest that telomere defects contribute to the pathogenesis of WS and perhaps other RecQ helicase diseases.     

Pathogen-induced production of the antifungal AFP protein from Aspergillus giganteus confers resistance to the blast fungus Magnaporthe grisea in transgenic rice

Rice blast, caused by Magnaporthe grisea, is the most important fungal disease of cultivated rice worldwide. We have developed a strategy for creating disease resistance to M. grisea whereby pathogen-induced expression of the afp (antifungal protein) gene from Aspergillus giganteus occurs in transgenic rice plants. Here, we evaluated the activity of the promoters from three maize pathogenesis-related (PR) genes, ZmPR4, mpi, and PRms, in transgenic rice. Chimeric gene fusions were prepared between the maize promoters and the beta-glucuronidase reporter gene (gus A). Histochemical assays of GUS activity in transgenic rice revealed that the ZmPR4 promoter is strongly induced in response to fungal infection, treatment with fungal elicitors, and mechanical wounding. The ZmPR4 promoter is not active in the seed endosperm. The mpi promoter also proved responsiveness to fungal infection and wounding but not to treatment with elicitors. In contrast, no activity of the PRms promoter in leaves of transgenic rice was observed. Transgenic plants expressing the afp gene under the control of the ZmPR4 promoter were generated. Transformants showed resistance to M. grisea at various levels. Our results suggest that pathogen-inducible expression of the afp gene in rice plants may be a practical way for protection against the blast fungus. Most agricultural crop species suffer from a vast array of fungal diseases that cause severe yield losses all over the world. Rice blast, caused by the fungus Magnaporthe grisea (Herbert) Barr (anamorph Pyricularia grisea), is the most devastating disease of cultivated rice (Oryza sativa L.), due to its     

A telomeric avirulence gene determines efficacy for the rice blast resistance gene Pi-ta

Genetic mapping showed that the rice blast avirulence gene AVR-Pita is tightly linked to a telomere on chromosome 3 in the plant pathogenic fungus Magnaporthe grisea. AVR-Pita corresponds in gene-for-gene fashion to the disease resistance (R) gene Pi-ta. Analysis of spontaneous avr-pita(-) mutants indicated that the gene is located in a telomeric 6.5-kb BglII restriction fragment. Cloning and DNA sequencing led to the identification of a candidate gene with features typical of metalloproteases. This gene is located entirely within the most distal 1.5 kb of the chromosome. When introduced into virulent rice pathogens, the cloned gene specifically confers avirulence toward rice cultivars that contain Pi-ta. Frequent spontaneous loss of AVR-Pita appears to be the result of its telomeric location. Diverse mutations in AVR-Pita, including point mutations, insertions, and deletions, permit the fungus to avoid triggering resistance responses mediated by Pi-ta. A point mutation in the protease consensus sequence abolishes the AVR-Pita avirulence function.     

OsBIRH1, a DEAD-box RNA helicase with functions in modulating defence responses against pathogen infection and oxidative stress

DEAD-box proteins comprise a large protein family with members from all kingdoms and play important roles in all types of processes in RNA metabolism. In this study, a rice gene OsBIRH1, which encodes a DEAD-box RNA helicase protein, was cloned and characterized. The predicted OsBIRH1 protein contains a DEAD domain and all conserved motifs that are common characteristics of DEAD-box RNA helicases. Recombinant OsBIRH1 protein purified from Escherichia coli was shown to have both RNA-dependent ATPase and ATP-dependent RNA helicase activities in vitro. Expression of OsBIRH1 was activated in rice seedling leaves after treatment with defence-related signal chemicals, for example benzothiadiazole, salicylic acid, l-aminocyclopropane-1-carboxylic acid, and jasmonic acid, and was also up-regulated in an incompatible interaction between a resistant rice genotype and the blast fungus, Magnaporthe grisea. Transgenic Arabidopsis plants that overexpress the OsBIRH1 gene were generated. Disease resistance phenotype assays revealed that the OsBIRH1-overexpressing transgenic plants showed an enhanced disease resistance against Alternaria brassicicola and Pseudomonas syringae pv. tomato DC3000. Meanwhile, defence-related genes, for example PR-1, PR-2, PR-5, and PDF1.2, showed an up-regulated expression in the transgenic plants. Moreover, the OsBIRH1 transgenic Arabidopsis plants also showed increased tolerance to oxidative stress and elevated expression levels of oxidative defence genes, AtApx1, AtApx2, and AtFSD1. The results suggest that OsBIRH1 encodes a functional DEAD-box RNA helicase and plays important roles in defence responses against biotic and abiotic stresses.     

Monomorphic subtelomeric DNA in the filamentous fungus, <Emphasis Type="Italic">Metarhizium anisopliae</Emphasis>,contains a RecQ helicase-like gene

In most filamentous fungi, telomere-associated sequences (TASs) are polymorphic, and the presence of restriction fragment length polymorphisms (RFLPs) may permit the number of chromosome ends to be estimated from the number of telomeric bands obtained by restriction digestion. Here, we describe strains of Metarhizium, Gliocladium and Paecilomyces species in which only one or a few telomeric bands of unequal intensity are detectable by Southern hybridization, indicating that interchromosomal TAS exchange occurs. We also studied an anomalous strain of Metarhizium anisopliae, which produces polymorphic telomeric bands larger than 8 kb upon digestion of genomic DNA with XhoI. In this case, the first XhoI site in from the chromosome end must lie beyond the presumed monomorphic region. Cloned telomeres from this strain comprise 18–26 TTAGGG repeats, followed at the internal end of the telomere tract by five repeats of the telomere-like sequence TAAACGCTGG. An 8.1-kb TAS clone also contains a gene for a RecQ-like helicase, designated TAH1, suggesting that this TAS is analogous to the Y elements in yeast and the subtelomeric helicase ORFs of Ustilago maydis (UTASRecQ) and Magnaporthe grisea (TLH1). The TAS in the anomalous strain of M. anisopliae, however, appears distinct from these in that it is found at most telomeres and its predicted protein product possesses a significantly longer N-terminal region in comparison to the M. grisea and U. maydis helicases. Hybridization analyses showed that TAH1 homologues are present in all other anomalous M. anisopliae strains studied, as well as in some other polymorphic strains, where the recQ-like gene also appears to be telomere-associated.     

稻瘟菌无毒基因研究进展

稻瘟菌是引起水稻稻瘟病的病原物。水稻与稻瘟菌间存在广泛而特异的相互作用,是研究寄主与病原物互作的重要模式系统。本文对稻瘟菌与水稻互作最重要的激发子―无毒基因的研究现状进行了概括,讨论了无毒基因的定位、克隆方法以及已克隆无毒基因的功能及进化研究,同时对今后无毒基因研究的重要方向进行了探讨,为深入理解无毒基因的功能及与水稻可能的互作关系奠定了基础。     

Genetic and Physical Mapping of Telomeres in the Rice Blast Fungus, Magnaporthe Grisea

Telomeric restriction fragments were genetically mapped to a previously described linkage map of Magnaporthe grisea, using RFLPs identified by a synthetic probe, (TTAGGG)(3). Frequent rearrangement of telomeric sequences was observed in progeny isolates creating a potential for misinterpretation of data. Therefore a consensus segregation data set was used to minimize mapping errors. Twelve of the 14 telomeres were found to be genetically linked to existing RFLP markers. Second-dimensional electrophoresis of restricted chromosomes confirmed these linkage assignments and revealed the chromosomal location of the two unlinked telomeres. We were thus able to assign all 14 M. grisea telomeres to their respective chromosome ends. The Achilles' cleavage (AC) technique was employed to determine that chromosome 1 markers 11 and CH5-120H were ~1.8 Mb and 1.28 Mb, respectively, from their nearest telomeres. RecA-AC was also used to determine that unlinked telomere 6 was ~530 kb from marker CH5-176H in strain 2539 and 580 kb in Guy11. These experiments indicated that large portions of some chromosome ends are unrepresented by genetic markers and provided estimates of the relationship of genetic to physical distance in these regions of the genome.     

MGOS: A resource for studying Magnaporthe grisea and Oryza sativa interactions

The MGOS (Magnaporthe grisea Oryza sativa) web-based database contains data from Oryza sativa and Magnaporthe grisea interaction experiments in which M. grisea is the fungal pathogen that causes the rice blast disease. In order to study the interactions, a consortium of fungal and rice geneticists was formed to construct a comprehensive set of experiments that would elucidate information about the gene expression of both rice and M. grisea during the infection cycle. These experiments included constructing and sequencing cDNA and robust long-serial analysis gene expression libraries from both host and pathogen during different stages of infection in both resistant and susceptible interactions, generating >50,000 M. grisea mutants and applying them to susceptible rice strains to test for pathogenicity, and constructing a dual O. sativa-M. grisea microarray. MGOS was developed as a central web-based repository for all the experimental data along with the rice and M. grisea genomic sequence. Community-based annotation is available for the M. grisea genes to aid in the study of the interactions.     

Analysis of genes expressed during rice-Magnaporthe grisea interactions.

Expressed sequence tag (EST) analysis was applied to identify rice genes involved in defense responses against infection by the blast fungus Magnaporthe grisea and fungal genes involved in growth within the host during a compatible interaction. A total of 511 clones was sequenced from a cDNA library constructed from rice leaves (Oryza sativa cv. Nipponbare) infected with M. grisea strain 70-15 to generate 296 nonredundant ESTs. The sequences of 293 clones (57.3%) significantly matched National Center for Biotechnology Information database entries; 221 showed homologies with previously identified plant genes and 72 with fungal genes. Among the genes with assigned functions, 32.8% were associated with metabolism, 29.4% with cell/organism defense or pathogenicity, and 18.4% with gene/protein expression. cDNAs encoding a type I metallothionein (MTs-1) of rice and a homolog of glucose-repressible gene 1 (GRG1) of Neurospora crassa were the most abundant representatives of plant and fungal genes, comprising 2.9 and 1.6% of the total clones, respectively. The expression patterns of 10 ESTs, five each from rice and M. grisea, were analyzed. Five defense-related genes in rice, including four pathogenesis-related genes and MTs-1, were highly expressed during M. grisea infection. Expression of five stress-inducible or pathogenicity-related genes of the fungus, including two hydrophobin genes, was also induced during growth within the host. Further characterization of the genes represented in this study would be an aid in unraveling the mechanisms of pathogenicity of M. grisea and the defense responses of rice.     

The IsTat 1.3 VSG multigene family in Trypanosoma brucei: retention of the expression linked copy through multiple antigenic switches.

In the IsTaR 1 serodeme of T. brucei the 3 variant surface glycoprotein (VSG) gene family contains about 10 members, one of which has a telomeric location on a minichromosome. The expression linked copy (ELC) of the 3 VSG gene which occurs in an antigenic variant expressing the 3 VSG, also has a telomeric location but unlike the minichromosomal 3 VSG gene has restriction sites upstream from the 5' barren region. This ELC is retained on the same telomere in a subsequent variant that expresses a telomeric 7 VSG ELC and in relapse variants and procyclic forms derived from variant antigenic types (VATs) 3 and 7. The 7 ELC has a restriction map upstream from the 5' barren region that differs from, but is similar to, that of the 3 ELC. These data indicate that the 3 and 7 ELCs are on different telomeres when expressed.     

The RecQ helicase AtRECQ4A is required to remove inter-chromosomal telomeric connections that arise during meiotic recombination in Arabidopsis

RecQ helicases are a conserved group of proteins with a role in the maintenance of genome integrity. In Saccharomyces cerevisiae (budding yeast), meiotic recombination is increased in the absence of the RecQ helicase Sgs1. Here we investigated the potential meiotic role of the Sgs1 homologue AtRECQ4A and the closely related AtRECQ4B. Both proteins have been shown to function during recombination in somatic cells, but so far their meiotic role has not been investigated. Both AtRECQ4A and AtRECQ4B were expressed in reproductive tissues. Although immunolocalization studies showed that AtRECQ4A associates with recombination intermediates, we found no evidence that its loss or that of AtRECQ4B had a significant effect on meiotic cross-overs, suggesting functional redundancy with other RECQ family members. Nevertheless, pollen viability decreased in Atrecq4A, resulting in a reduction in fertility, although this was not the case in Atrecq4B. Cytological analysis revealed chromatin bridges between the telomeres of non-homologous chromosomes in Atrecq4A at metaphase I, in some instances accompanied by chromosome fragmentation at anaphase I. The bridges required telomeric repeats and were dependent on meiotic recombination. Immunolocalization confirmed the association of AtRECQ4A with the telomeres during prophase I, which we propose enables dissolution of recombination-dependent telomeric associations. Thus, this study has identified a hitherto unknown role for a member of the RECQ helicase family during meiosis that contributes to the maintenance of chromosome integrity. As telomere structure is generally conserved, it seems likely that these associations may arise during meiosis in other species, where they must also be removed.     

Germline mutations of regulator of telomere elongation helicase 1, RTEL1, in Dyskeratosis congenita

Dyskeratosis congenita (DC) is an inherited bone marrow failure and cancer predisposition syndrome caused by aberrant telomere biology. The classic triad of dysplastic nails, abnormal skin pigmentation, and oral leukoplakia is diagnostic of DC, but substantial clinical heterogeneity exists; the clinically severe variant Hoyeraal Hreidarsson syndrome (HH) also includes cerebellar hypoplasia, severe immunodeficiency, enteropathy, and intrauterine growth retardation. Germline mutations in telomere biology genes account for approximately one-half of known DC families. Using exome sequencing, we identified mutations in RTEL1, a helicase with critical telomeric functions, in two families with HH. In the first family, two siblings with HH and very short telomeres inherited a premature stop codon from their mother who has short telomeres. The proband from the second family has HH and inherited a premature stop codon in RTEL1 from his father and a missense mutation from his mother, who also has short telomeres. In addition, inheritance of only the missense mutation led to very short telomeres in the proband’s brother. Targeted sequencing identified a different RTEL1 missense mutation in one additional DC proband who has bone marrow failure and short telomeres. Both missense mutations affect the helicase domain of RTEL1, and three in silico prediction algorithms suggest that they are likely deleterious. The nonsense mutations both cause truncation of the RTEL1 protein, resulting in loss of the PIP box; this may abrogate an important protein–protein interaction. These findings implicate a new telomere biology gene, RTEL1, in the etiology of DC.     

转拟南芥AtNPR1基因增强水稻对水稻白叶枯病和稻瘟病的抗性

AtNPR1基因是拟南芥系统获得抗性的一个重要调节基因,在拟南芥中过量表达AtNPR1基因能使拟南芥对细菌和真菌的抗性同时增强.为了研究在水稻中过量表达AtNPR1基因对水稻抗病性的影响,将该基因转入到广西主栽籼稻恢复系品种桂99中.经PCR验证得到了79株转基因植株,DNA斑点杂交表明ATNPR1基因已经整合到桂99染色体DNA中.Northern杂交和RT-PCR分析表明,AtNPR1基因在桂99中已经表达;同时还检测了转基因植株对水稻白叶枯病和稻瘟病的抗性,结果表明转基因植株对该两种病害的抗性均显著增强.     

The Magnaporthe grisea snodprot1 homolog, MSP1, is required for virulence

Secreted proteins play central roles in plant-microbe interactions acting as signals, toxins, and effectors. One important group of small secreted proteins is the snodprot1 family, members of which have demonstrated phytotoxic properties. A split-marker transformation system was applied for gene deletion of the snodprot1 homolog, MSP1, in the rice blast fungus Magnaporthe grisea. msp1 mutants were phenotypically indistinguishable from wild type and elaborated apparently normal appressoria. However, the deletion mutants were greatly reduced in virulence primarily due to impaired growth in planta. Western blot analysis showed that the protein was secreted and not associated with the fungal cell wall. When purified MSP1 protein was applied to wounded leaf tissue, no apparent phytotoxic effects were noted. This is the first report to the authors' knowledge that directly implicates a snodprot1 protein as a virulence factor.     

Telomere-targeted retrotransposons in the rice blast fungus Magnaporthe oryzae: agents of telomere instability

The fungus Magnaporthe oryzae is a serious pathogen of rice and other grasses. Telomeric restriction fragments in Magnaporthe isolates that infect perennial ryegrass (prg) are hotspots for genomic rearrangement and undergo frequent, spontaneous alterations during fungal culture. The telomeres of rice-infecting isolates are very stable by comparison. Sequencing of chromosome ends from a number of prg-infecting isolates revealed two related non-LTR retrotransposons (M. oryzae Telomeric Retrotransposons or MoTeRs) inserted in the telomere repeats. This contrasts with rice pathogen telomeres that are uninterrupted by other sequences. Genetic evidence indicates that the MoTeR elements are responsible for the observed instability. MoTeRs represent a new family of telomere-targeted transposons whose members are found exclusively in fungi.     

Strand exchange of telomeric DNA catalyzed by the Werner syndrome protein (WRN) is specifically stimulated by TRF2

Werner syndrome (WS), caused by loss of function of the RecQ helicase WRN, is a hereditary disease characterized by premature aging and elevated cancer incidence. WRN has DNA binding, exonuclease, ATPase, helicase and strand annealing activities, suggesting possible roles in recombination-related processes. Evidence indicates that WRN deficiency causes telomeric abnormalities that likely underlie early onset of aging phenotypes in WS. Furthermore, TRF2, a protein essential for telomere protection, interacts with WRN and influences its basic helicase and exonuclease activities. However, these studies provided little insight into WRN''s specific function at telomeres. Here, we explored the possibility that WRN and TRF2 cooperate during telomeric recombination processes. Our results indicate that TRF2, through its interactions with both WRN and telomeric DNA, stimulates WRN-mediated strand exchange specifically between telomeric substrates; TRF2''s basic domain is particularly important for this stimulation. Although TRF1 binds telomeric DNA with similar affinity, it has minimal effects on WRN-mediated strand exchange of telomeric DNA. Moreover, TRF2 is displaced from telomeric DNA by WRN, independent of its ATPase and helicase activities. Together, these results suggest that TRF2 and WRN act coordinately during telomeric recombination processes, consistent with certain telomeric abnormalities associated with alteration of WRN function.     

Family 19 chitinase of Streptomyces griseus HUT6037 increases plant resistance to the fungal disease

Chitinase C (ChiC) is the first bacterial family 19 chitinase discovered in Streptomyces griseus HUT6037. In vitro, ChiC clearly inhibited hyphal extension of Trichoderma reesei but a rice family 19 chitinase did not. In order to investigate the effects of ChiC as an increaser of plant resistance to fungal diseases, the chiC gene was introduced into rice plants under the control of the increased CaMV 35S promoter and a signal sequence from the rice chitinase gene. Transgenic plants were morphologically normal. Resistance to leaf blast disease caused by Magnaporthe grisea was evaluated in R1 and R2 generations using a spray method. Ninety percent of transgenic rice plants expressing ChiC had higher resistance than non-transgenic plants. Disease resistance of sibling plants within the same line was correlated with the ChiC expression levels. ChiC produced in rice plants accumulated intercellularly and had the hydrolyzing activity against glycol chitin.