Characterization of the BLR1 gene encoding a putative blue-light regulator in the phytopathogenic fungus Bipolaris oryzae

Bipolaris oryzae is a filamentous ascomycetous fungus that causes brown leaf spot disease in rice. We isolated and characterized BLR1, a gene that encodes a putative blue-light regulator similar to Neurospora crassa white-collar 1 (WC-1). The deduced amino acid sequence of BLR1 showed high degrees of similarity to other fungal blue-light regulator protein. Disruption of the BLR1 gene demonstrated that this gene is essential for conidial development after conidiophore formation and for near-UV radiation-enhanced photolyase gene expression.     

A Pmk1-interacting gene is involved in appressorium differentiation and plant infection in Magnaporthe oryzae

In the rice blast fungus Magnaporthe oryzae, the PMK1 mitogen-activated protein (MAP) kinase gene regulates appressorium formation and infectious growth. Its homologs in many other fungi also play critical roles in fungal development and pathogenicity. However, the targets of this important MAP kinase and its interacting genes are not well characterized. In this study, we constructed two yeast two-hybrid libraries of M. oryzae and screened for Pmk1-interacting proteins. Among the nine Pmk1-interacting clones (PICs) identified, two of them, PIC1 and PIC5, were selected for further characterization. Pic1 has one putative nuclear localization signal and one putative MAP kinase phosphorylation site. Pic5 contains one transmembrane domain and two functionally unknown CTNS (cystinosin/ERS1p repeat) motifs. The interaction of Pmk1 with Pic1 or Pic5 was confirmed by coimmunoprecipitation assays. Targeted gene deletion of PIC1 had no apparent effects on vegetative growth and pathogenicity but resulted in a significant reduction in conidiation and abnormal germ tube differentiation on onion epidermal cells. Deletion of PIC5 led to a reduction in conidiation and hyphal growth. Autolysis of aerial hyphae became visible in cultures older than 4 days. The pic5 mutant was defective in germ tube growth and appressorium differentiation. It was reduced in appressorial penetration and virulence on the plant. Both PIC1 and PIC5 are conserved in filamentous ascomycetes, but none of their orthologs have been functionally characterized. Our data indicate that PIC5 is a novel virulence factor involved in appressorium differentiation and pathogenesis in M. oryzae.     

Nucleotide sequence of a genomic and a cDNA clone encoding an extracellular alkaline protease of Aspergillus fumigatus

An Aspergillus fumigatus extracellular alkaline protease (ALP) which is an enzyme of the subtilisin family is a potential virulent factor of the fungus. The gene encoding ALP was isolated from a genomic library made from DNA of an A. fumigatus isolate. The nucleotide sequence of this gene was compared to that of a cDNA encoding A. oryzae ALP and to that of a cDNA from A. fumigatus encoding the mature ALP protein. Mature A. fumigatus ALP contains 282 amino acids and is encoded by three exons. The pre-proenzyme has a leader sequence of 121 amino acids.     

RNA-mediated gene silencing in the phytopathogenic fungus Bipolaris oryzae

The Ascomycetous fungus Bipolaris oryzae is the causal agent of brown leaf spot disease in rice and is a model for studying photomorphogenetic responses by near-UV radiation. Targeted gene disruption (knockout) for functional analysis of photomorphogenesis-related genes in B. oryzae can be achieved by homologous recombination with low efficiency. Here, the applicability of RNA silencing (knockdown) as a tool for targeting endogenous genes in B. oryzae is reported. A polyketide synthase gene (PKS1), involved in fungal DHN melanin biosynthesis pathways, was targeted by gene silencing as a marker. The silencing vector encoding hairpin RNA of the PKS1 fragment was constructed in a two-step PCR-based cloning, and introduced into the B. oryzae genomic DNA. Silencing of the PKS1 gene resulted in albino phenotypes and reduction of PKS1 mRNA expression. These results demonstrate the applicability of targeted gene silencing as a useful reverse-genetics approach in B. oryzae.     

血红密孔菌(Pycnoporussanguineus)漆酶基因的克隆与序列分析

为克隆血红密孔菌 (Pycnoporussanguineus)漆酶基因 ,根据真菌漆酶氨基酸序列保守区设计了 1对简并引物 .以血红密孔菌基因组DNA为模板 ,PCR扩增出长 12 2 7bp的漆酶基因片段 .以此序列为基础 ,通过 5′及 3′RACE技术克隆出漆酶全长cDNA序列 ,序列长为 190 2bp ,其 5′端和 3′端非编码区长分别为 5 1bp和 2 97bp ,开放阅读框长 15 5 4bp ,编码 5 18个氨基酸的蛋白 .该蛋白具有 4个铜离子结合区域 ,预测其相对分子量为 5 6 313 2 ,等电点为 5 5 9,其氨基酸序列与Pycnoporuscinnabarinus漆酶 (lcc3 2 )的同源性最高 ,为 96 % .以该cDNA编码区的两端序列为引物 ,PCR扩增得到漆酶的长度为 2 15 4bp的全长DNA序列 ,序列中包括 10个内含子序列 ,长为 5 2～ 70bp     

Cloning and characterization of vmaA,the gene encoding a 69-kDa catalytic subunit of the vacuolar H+-ATPase during alkaline pH mediated growth of Aspergillus oryzae

Screening of a cDNA library constructed under alkaline pH mediated growth of Aspergillus oryzae implicated a vacuolar H+-ATPase gene (vmaA) as a putative candidate involved in alkaline pH adaptation. A. oryzae vmaA genomic DNA extended to 2072 bp including three introns and encoded a protein of 605 amino acids. VmaAp was homologous to Vma-1p from Neurospora crassa (71%), Vma1p from Saccharomyces cerevisiae (69%) and ATP6A2 from human (49%). The vmaA cDNA complemented S. cerevisiae V-ATPase disrupted strain (Deltavma1) was viable at alkaline pH 8.0 and in the presence of CaCl(2) (100 mM). Northern analysis revealed an enhanced expression of vmaA during growth of A. oryzae in alkaline medium (pH 10.0). The A. oryzae vmaA disruptant exhibited abnormally shrunken vacuoles and hyphal walls at pH 8.5 and a growth defect at pH 10.0, implicating an alkaline pH stress responsive role for vmaA in A. oryzae.     

Nucleotide sequence and expression of the glucoamylase-encoding gene (glaA) from Aspergillus oryzae.

The glucoamylase-encoding gene (glaA) from Aspergillus oryzae was cloned using its cDNA as a probe, which had been isolated previously. From comparison of nucleotide (nt) sequences of genomic clones with its cDNA, the glaA gene was found to contain four short putative introns, 45-56 nt in length. The A. oryzae glaA gene shared 62% homology at the nt level with the A. niger glaA gene with the four introns located at the same position. The 5'-flanking region contained a TATA box at nt-72 from the start codon, and two putative CAAT sequences at nt-87 and -331. Genomic Southern analysis and physical mapping showed that the glaA gene is located on the smallest chromosome (3.4 Mb) of six separated bands of chromosomes. Clones containing the glaA gene, when re-introduced intro A. oryzae, resulted in a three- to eightfold increase in glucoamylase activity.     

Cloning and characterization of genes specifically expressed during infection stages in the rice blast fungus

We isolated promoters of 12 genes from the rice blast fungus based on the sequences of randomly selected expressed sequence tags (ESTs) (appressorium formation stage cDNA library of Magnaporthe available from GenBank). These promoters (and the 5' coding regions if any) were fused in frame with egfp, and their expression patterns were examined under the epifluorescence microscope. Among them, two turned out to be specifically active in structures necessary for infection, viz. a promoter of adenylate cyclase interacting protein 1-like gene expressed in conidia, germ tubes, and appressoria, and a promoter of putative membrane-associated or secreted protein gene specifically expressed in appressoria. Although targeted knockout mutants of either gene failed to show detectable phenotypic alterations under laboratory conditions, these ESTs should be useful for identification of genes expressed during infection stages.     

Novel genomic locus with atypical G+C content that is required for extracellular polysaccharide production and virulence in Xanthomonas oryzae pv. oryzae.

Three exopolysaccharide (EPS)- and virulence-deficient mutants of Xanthomonas oryzae pv. oryzae, the causal agent of bacterial leaf blight of rice, were isolated by Tn5 mutagenesis. These insertions are not located within the gum gene cluster. A 40-kb cosmid clone that restored EPS production and virulence to all three mutants was isolated, and the three transposon insertions were localized to contiguous 4.3- and 3.5-kb EcoRI fragments that are included in this clone. Sequence data indicate that two of the transposon insertions are in genes that encode a putative sugar nucleotide epimerase and a putative glycosyl transferase, respectively; the third insertion is located between the glycosyl transferase gene and a novel open reading frame (ORF). A 5.5-kb genomic region in which these three ORFs are located has a G+C content of 5-1.7%, quite different from the G+C content of approximately 65.0% that is typical of X. oryzae pv. oryzae. Homologues of this locus have not yet been reported in any other xanthomonad.     

Isolation and characterization of a cDNA from Trichoderma harzianum P1 encoding a 14-3-3 protein homolog

A full-length cDNA clone, Th1433, (GenBank accession No. U24158), was isolated and characterized from the filamentous fungus, Trichoderma harzianum. The deduced amino acid (aa) sequence showed an acidic 30-kDa protein homologous to the 14-3-3 proteins, a family of putative kinase regulators originally characterized in mammalian brain tissue. The greatest homology, 71% identical aa, was found to BMH1, the corresponding protein from Saccharomyces cerevisiae and to the ε isoform from sheep brain. Southern analysis of genomic DNA indicated that Th1433 is a member of a small genomic family. At least two genes encoding 14-3-3-like proteins exist in T. harzianum. Northern analysis showed the highest level of expression during the first day after inoculation of the culture with conidial spores.