Maintenance of Sex-Related Genes and the Co-Occurrence of Both Mating Types in Verticillium dahliae

Verticillium dahliae is a cosmopolitan, soilborne fungus that causes a significant wilt disease on a wide variety of plant hosts including economically important crops, ornamentals, and timber species. Clonal expansion through asexual reproduction plays a vital role in recurring plant epidemics caused by this pathogen. The recent discovery of recombination between clonal lineages and preliminary investigations of the meiotic gene inventory of V. dahliae suggest that cryptic sex appears to be rare in this species. Here we expanded on previous findings on the sexual nature of V. dahliae. Only 1% of isolates in a global collection of 1120 phytopathogenic V. dahliae isolates contained the MAT1-1 idiomorph, whereas 99% contained MAT1-2. Nine unique multilocus microsatellite types comprised isolates of both mating types, eight of which were collected from the same substrate at the same time. Orthologs of 88 previously characterized sex-related genes from fungal model systems in the Ascoymycota were identified in the genome of V. dahliae, out of 93 genes investigated. Results of RT-PCR experiments using both mating types revealed that 10 arbitrarily chosen sex-related genes, including MAT1-1-1 and MAT1-2-1, were constitutively expressed in V. dahliae cultures grown under laboratory conditions. Ratios of non-synonymous (amino-acid altering) to synonymous (silent) substitutions in V. dahliae MAT1-1-1 and MAT1-2-1 sequences were indistinguishable from the ratios observed in the MAT genes of sexual fungi in the Pezizomycotina. Patterns consistent with strong purifying selection were also observed in 18 other arbitrarily chosen V. dahliae sex-related genes, relative to the patterns in orthologs from fungi with known sexual stages. This study builds upon recent findings from other laboratories and mounts further evidence for an ancestral or cryptic sexual stage in V. dahliae.     

Genome-Wide Identification,Phylogeny and Expression Profile of Vesicle Fusion Components in Verticillium dahliae

Vesicular trafficking plays a crucial role in protein localization and movement, signal transduction, and multiple developmental processes in eukaryotic cells. Vesicle fusion is the final and key step in vesicle-mediated trafficking and mainly relies on SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors), the regulators including SM (Sec1/Munc18) family proteins, Rab GTPases and exocyst subunits. Verticillium dahliae is a widespread soil fungus that causes disruptive vascular diseases on a wide range of plants. To date, no genes involved in vesicular fusion process have been identified and characterized in V. dahliae. The recent publication of the draft genome sequence of V. dahliae allowed us to conduct a genome-wide identification, phylogeny and expression profile of genes encoding vesicular fusion components. Using compared genomics and phylogenetic methods, we identified 44 genes encoding vesicle fusion components in the V. dahliae genome. According to the structural features of their encoded proteins, the 44 V. dahliae genes were classified into 22 SNAREs (6 Qa-, 4 Qb-, 6 Qc-, 1 Qbc- and 5 R-types), 4 SM family proteins, 10 Rab GTPases and 8 exocyst proteins. Based on phylogeny and motif constitution analysis, orthologs of vesicle fusion component in filamentous fungi were generally clustered together into the same subclasses with well-supported bootstrap values. Analysis of the expression profiles of these genes indicated that many of them are significantly differentially expressed during vegetative growth and microsclerotia formation in V. dahliae. The analysis show that many components of vesicle fusion are well conserved in filamentous fungi and indicate that vesicle fusion plays a critical role in microsclerotia formation of smoke tree wilt fungus V. dahliae. The genome-wide identification and expression analysis of components involved in vesicle fusion should facilitate research in this gene family and give new insights toward elucidating their functions in growth, development and pathogenesis of V. dahliae.     

The aux1 Mutation of Arabidopsis Confers Both Auxin and Ethylene Resistance

Mutagenized populations of Arabidopsis thaliana seedlings were screened for plants capable of root growth on inhibitory concentrations of the ethylene precursor 1-aminocyclopropane-1-carboxylic acid. Four of the mutant lines recovered from this screen display a defect in root gravitropism as well as hormone resistance. The aerial portions of these plants are similar to wild-type in appearance. Genetic analysis of these four mutants demonstrated that hormone resistance segregated as a recessive trait and that all four mutations were alleles of the auxin-resistant mutation aux1 [Maher HP, Martindale SJB (1980) Biochem Genet 18: 1041-1053]. These new mutants have been designated aux1-7, 1-12, 1-15, and 1-19. The sensitivity of wild-type and aux1-7 roots to indole-3-acetic acid, 2,4-dichlorophenoxyacetic acid, and ethylene was determined. The results of these assays show that aux1-7 plants require a 12-fold (indole-3-acetic acid) or 18-fold (2,4-dichlorophenoxyacetic acid) higher concentration of auxin than wild-type for a 50% inhibition of root growth. In addition, ethylene inhibition of root growth in aux1-7 plants is approximately 30% that of wild-type at saturating ethylene concentrations. These results indicate that aux1 plants are resistant to both auxin and ethylene. We have also determined the effect of ethylene treatment on chlorophyll loss and peroxidase activity in the leaves of aux1 and wild-type plants. No difference between mutant and wild-type plants was observed in these experiments, indicating that hormone resistance in aux1 plants may be limited to root growth. Our studies suggest that the AUX1 gene may have a specific function in the hormonal regulation of gravitropism.     

Variation in sic Gene Encoding Complement-Inhibiting Protein of Streptococcus pyogenes Serotype M1 Isolates in Japan

DNA sequencing of the gene encoding a complement-inhibiting protein of Streptococcus pyogenes (streptococcal inhibitor of complement, Sic) was carried out on 49 strains of S. pyogenes serotype M1. Those strains were obtained from patients and asymptomatic carriers in Japan from 1969 to 1997 and had various pulsed-field gel electrophoresis (PFGE) patterns. Four identical polymorphic sites were found in the strains with the same PFGE pattern (Ia), but not in those giving the pattern IIa. The other identical sites were found in the strains with the PFGE pattern IIa, but not in those with the pattern Ia. These observations suggest that each of PFGE patterns was restricted to a set of variation in the sic gene. Received: 13 January 2000 / Accepted: 24 February 2000     

Molecular Mapping and Validation of a Major QTL Conferring Resistance to a Defoliating Isolate of Verticillium Wilt in Cotton (Gossypium hirsutum L.)

Verticillium wilt (VW) caused by Verticillium dahliae Kleb is one of the most destructive diseases of cotton. Development and use of a VW resistant variety is the most practical and effective way to manage this disease. Identification of highly resistant genes/QTL and the underlining genetic architecture is a prerequisite for developing a VW resistant variety. A major QTL qVW-c6-1 conferring resistance to the defoliating isolate V991 was identified on chromosome 6 in LHB22×JM11 F_2∶3 population inoculated and grown in a greenhouse. This QTL was further validated in the LHB22×NNG F_2∶3 population that was evaluated in an artificial disease nursery of V991 for two years and in its subsequent F₄ population grown in a field severely infested by V991. The allele conferring resistance within the QTL qVW-c6-1 region originated from parent LHB22 and could explain 23.1–27.1% of phenotypic variation. Another resistance QTL qVW-c21-1 originated from the susceptible parent JM11 was mapped on chromosome 21, explaining 14.44% of phenotypic variation. The resistance QTL reported herein provides a useful tool for breeding a cotton variety with enhanced resistance to VW.     

Characterization of a Novel Cotton Subtilase Gene GbSBT1 in Response to Extracellular Stimulations and Its Role in Verticillium Resistance

Verticillium wilt is a disastrous vascular disease in plants caused by Verticillium dahliae. Verticillium pathogens secrete various disease-causing effectors in cotton. This study identified a subtilase gene GbSBT1 from Gossypium babardense and investigated the roles against V. dahliae infection. GbSBT1 gene expression is responsive to V. dahliae defense signals, jasmonic acid, and ethylene treatments. Moreover, the GbSBT1 protein is mainly localized in the cell membrane and moves into the cytoplasm following jasmonic acid and ethylene treatments. Silencing GbSBT1 gene expression through virus-induced GbSBT1 gene silencing reduced the tolerance of Pima-90 (resistant genotype), but not facilitated the infection process of V. dahliae in Coker-312 (sensitive genotype). Moreover, the ectopically expressed GbSBT1 gene enhanced the resistance of Arabidopsis to Fusarium oxysporum and V. dahliae infection and activated the expression levels of defense-related genes. Furthermore, pull-down, yeast two-hybrid assay, and BiFC analysis revealed that GbSBT1 interacts with a prohibitin (PHB)-like protein expressed in V. dahliae pathogens during infection. In summary, GbSBT1 recognizes the effector PHB protein secreted from V. dahliae and is involved in Verticillium-induced resistance in cotton.     

Ectopic Expression of Rice OsBIANK1, Encoding an Ankyrin Repeat‐Containing Protein,in Arabidopsis Confers Enhanced Disease Resistance to Botrytis cinerea and Pseudomonas syringae

Ankyrin repeat‐containing proteins comprise a large family whose members have been shown to play important roles in various aspects of biological processes in plant growth and development as well as in responses to biotic and abiotic stresses. We previously identified a rice gene, OsBIANK1, encoding an ankyrin repeat‐containing protein and found that expression of OsBIANK1 can be induced by defence signalling molecules and by infection of Magnaporthe oryzae, the causal agent of blast disease. To better understand the possible function of OsBIANK1 in disease resistance, we generated transgenic Arabidopsis plants that constitutively overexpress the OsBIANK1 gene. Results from disease assays revealed that the OsBIANK1‐overexpressing plants display increased resistance against Botrytis cinerea and Pseudomonas syringae pv. tomato DC3000 as compared with the wild‐type plants. In OsBIANK1‐overexpressing plants, expression of some of well‐known defence genes (e.g. PR‐1, PR‐2 and PDF1.2) was up‐regulated after infection with B. cinerea or P. syringae pv. tomato DC3000. Furthermore, the OsBIANK1‐overexpressing plants showed decreased levels of reactive oxygen species (i.e. superoxide anion and H₂O₂) after Botrytis infection. Thus, our present results further support the role of OsBIANK1 in regulation of defence responses against different types of pathogens.     

Perturbations in the Primary Metabolism of Tomato and Arabidopsis thaliana Plants Infected with the Soil-Borne Fungus Verticillium dahliae

The hemibiotrophic soil-borne fungus Verticillium dahliae is a major pathogen of a number of economically important crop species. Here, the metabolic response of both tomato and Arabidopsis thaliana to V. dahliae infection was analysed by first using non-targeted GC-MS profiling. The leaf content of both major cell wall components glucuronic acid and xylose was reduced in the presence of the pathogen in tomato but enhanced in A. thaliana. The leaf content of the two tricarboxylic acid cycle intermediates fumaric acid and succinic acid was increased in the leaf of both species, reflecting a likely higher demand for reducing equivalents required for defence responses. A prominent group of affected compounds was amino acids and based on the targeted analysis in the root, it was shown that the level of 12 and four free amino acids was enhanced by the infection in, respectively, tomato and A. thaliana, with leucine and histidine being represented in both host species. The leaf content of six free amino acids was reduced in the leaf tissue of diseased A. thaliana plants, while that of two free amino acids was raised in the tomato plants. This study emphasizes the role of primary plant metabolites in adaptive responses when the fungus has colonized the plant.     

Over-Expression of Cysteine Leucine Rich Protein Is Related to SAG Resistance in Clinical Isolates of Leishmania donovani

Background

Resistance emergence against antileishmanial drugs, particularly Sodium Antimony Gluconate (SAG) has severely hampered the therapeutic strategy against visceral leishmaniasis, the mechanism of resistance being indistinguishable. Cysteine leucine rich protein (CLrP), was recognized as one of the overexpressed proteins in resistant isolates, as observed in differential proteomics between sensitive and resistant isolates of L. donovani. The present study deals with the characterization of CLrP and for its possible connection with SAG resistance.

Methodology and Principal Findings

In pursuance of deciphering the role of CLrP in SAG resistance, gene was cloned, over-expressed in E. coli system and thereafter antibody was raised. The expression profile of CLrP and was found to be over-expressed in SAG resistant clinical isolates of L. donovani as compared to SAG sensitive ones when investigated by real-time PCR and western blotting. CLrP has been characterized through bioinformatics, immunoblotting and immunolocalization analysis, which reveals its post-translational modification along with its dual existence in the nucleus as well as in the membrane of the parasite. Further investigation using a ChIP assay confirmed its DNA binding potential. Over-expression of CLrP in sensitive isolate of L. donovani significantly decreased its responsiveness to SAG (SbV and SbIII) and a shift towards the resistant mode was observed. Further, a significant increase in its infectivity in murine macrophages has been observed.

Conclusion/Significance

The study reports the differential expression of CLrP in SAG sensitive and resistant isolates of L. donovani. Functional intricacy of CLrP increases with dual localization, glycosylation and DNA binding potential of the protein. Further over-expressing CLrP in sensitive isolate of L. donovani shows significantly decreased sensitivity towards SAG and increased infectivity as well, thus assisting the parasite in securing a safe niche. Results indicates the possible contribution of CLrP to antimonial resistance in L. donovani by assisting the parasite growth in the macrophages.     

Mos1-Mediated Transgenesis to Probe Consequences of Single Gene Mutations in Variation-Rich Isolates of Caenorhabditis elegans

Caenorhabditis elegans, especially the N2 isolate, is an invaluable biological model system. Numerous additional natural C. elegans isolates have been shown to have unexpected genotypic and phenotypic variations which has encouraged researchers to use next generation sequencing methodology to develop a more complete picture of genotypic variations among the isolates. To understand the phenotypic effects of a genomic variation (GV) on a single gene, in a variation-rich genetic background, one should analyze that particular GV in a well understood genetic background. In C. elegans, the analysis is usually done in N2, which requires extensive crossing to bring in the GV. This can be a very time consuming procedure thus it is important to establish a fast and efficient approach to test the effect of GVs from different isolates in N2. Here we use a Mos1-mediated single-copy insertion (MosSCI) method for phenotypic assessments of GVs from the variation-rich Hawaiian strain CB4856 in N2. Specifically, we investigate effects of variations identified in the CB4856 strain on tac-1 which is an essential gene that is necessary for mitotic spindle elongation and pronuclear migration. We show the usefulness of the MosSCI method by using EU1004 tac-1(or402) as a control. or402 is a temperature sensitive lethal allele within a well-conserved TACC domain (transforming acidic coiled-coil) that results in a leucine to phenylalanine change at amino acid 229. CB4856 contains a variation that affects the second exon of tac-1 causing a cysteine to tryptophan change at amino acid 94 also within the TACC domain. Using the MosSCI method, we analyze tac-1 from CB4856 in the N2 background and demonstrate that the C94W change, albeit significant, does not cause any obvious decrease in viability. This MosSCI method has proven to be a rapid and efficient way to analyze GVs.     

棉花抗逆相关基因GhDr1的克隆及生物信息学分析

通过TAIL-PCR的方法从棉花中克隆到未知功能的新基因GhDr1,生物信息学分析结果表明,GhDr1的预测蛋白含有酪氨酸激酶和蛋白酶C磷酸化位点、潜在的锌指类功能模块、MAPK磷酸化位点及MAPK相互作用识别位点;与GhDr1同源基因位于同一基因簇的基因多为参与逆境信号转导的蛋白。推测GhDr1基因可能在逆境信号转导途径的磷酸化级联反应中被调节,参与棉花逆境应答的生理过程。     

Complementation of the Arabidopsis pds1 Mutation with the Gene Encoding p-Hydroxyphenylpyruvate Dioxygenase

Plastoquinone and tocopherols are the two major quinone compounds in higher plant chloroplasts and are synthesized by a common pathway. In previous studies we characterized two loci in Arabidopsis defining key steps of this biosynthetic pathway. Mutation of the PDS1 locus disrupts the activity of p-hydroxyphenylpyruvate dioxygenase (HPPDase), the first committed step in the synthesis of both plastoquinone and tocopherols in plants. Although plants homozygous for the pds1 mutation could be rescued by growth in the presence of homogentisic acid, the product of HPPDase, we were unable to determine if the mutation directly or indirectly disrupted HPPDase activity. This paper reports the isolation of a cDNA, pHPPD, encoding Arabidopsis HPPDase and its functional characterization by expression in both plants and Escherichia coli. pHPPD encodes a 50-kD polypeptide with homology to previously identified HPPDases, including 37 highly conserved amino acid residues clustered in the carboxyl region of the protein. Expression of pHPPD in E. coli catalyzes the accumulation of homogentisic acid, indicating that it encodes a functional HPPDase enzyme. Mapping of pHPPD and co-segregation analysis of the pds1 mutation and the HPPD gene indicate tight linkage. Constitutive expression of pHPPD in a pds1 mutant background complements this mutation. Finally, comparison of the HPPD genomic sequences from wild type and pds1 identified a 17-bp deletion in the pds1 allele that results in deletion of the carboxyterminal 26 amino acids of the HPPDase protein. Together, these data conclusively demonstrate that pds1 is a mutation in the HPPDase structural gene.     

Theoretical Model of the Three-dimensional Structure of a Disease Resistance Gene Homolog Encoding Resistance Protein in Vigna mungo

Abstract

Plant disease resistance (R) genes, the key players of innate immunity system in plants encode ‘R’ proteins. ‘R’ protein recognizes product of avirulance gene from the pathogen and activate downstream signaling responses leading to disease resistance. No three dimensional (3D) structural information of any ‘R’ proteins is available as yet. We have reported a ‘R’ gene homolog, the 'VMYR1′, encoding ‘R’ protein in Vigna mungo. Here, we describe the homology modeling of the 'VMYR1′ protein. The model was created by using the 3D structure of an ATP-binding cassette transporter protein from Vibrio cholerae as a template. The strategy for homology modeling was based on the high structural conservation in the superfamily of P-loop containing nucleoside triphosphate hydrolase in which target and template proteins belong. This is the first report of theoretical model structure of any ‘R’ proteins.     

Resistance of Cotton to the Root-Knot Nematode, Meloidogyne incognita

Cotton plants resistant to Meloidogyne incognita had roots characterized by fewer and smaller galls, and females that produced fewer egg masses containing fewer eggs than did susceptible plants. Many galls on resistant roots contained no nematodes at the time of examination. Penetration of the resistant cultivar was equal to that of the susceptible cultivar and independent of the number of nematodes in the inoculum. Fewer nematodes penetrated resistant or susceptible plants with eight leaves than those with fewer leaves. Reciprocal grafts of resistant and susceptible plants failed to confer resistance or susceptibility to the rootstock.