Two flagellar stators and their roles in motility and virulence in <Emphasis Type="Italic">Pseudomonas syringae</Emphasis> pv. <Emphasis Type="Italic">tabaci</Emphasis> 6605

The motor proteins around the flagellar basal body consist of two cytoplasmic membrane proteins, MotA and MotB, and function as a complex that acts as the stator to generate the torque that drives rotation. Genome analysis of several Pseudomonas syringae pathovars revealed that there are two sets of genes encoding motor proteins: motAB and motCD. Deduced amino acid sequences for MotA/B and MotC/D showed homologies to the H⁺-driven stator from Escherichia coli and Na⁺-driven stator from Vibrio alginolyticus, respectively. However, the swimming motility of P. syringae pv. tabaci (Pta) 6605 was inhibited by the protonophore carbonyl cyanide m-chlorophenylhydrazone but not by the sodium stator-specific inhibitor phenamil. To identify a gene encoding the stator protein required for motility, ∆motAB, ∆motCD, and ∆motABCD mutants were generated. The ∆motCD mutant had remarkably reduced and the ∆motABCD mutant completely abolished swimming motilities, whereas the ∆motAB mutant retained some degree of these abilities. The ∆motCD and ∆motABCD mutants did not produce N-acyl-homoserine lactones (AHLs), quorum-sensing molecules in this pathogen, and remarkably reduced the ability to cause disease in host tobacco leaves, as we previously observed in the ∆fliC mutant strain. These results strongly indicate that both stator pairs in Pta 6605 are proton-dependent and that MotCD is important for not only flagellar motility but also for production of AHLs and the ability to cause disease in host plants.     

Proton Motive Force in <Emphasis Type="Italic">Rhodobacter sphaeroides</Emphasis> Under Anaerobic Conditions in the Dark

In order to examine the mediatory role of proton motive force (∆p) or proton ATPase in H₂ production by Rhodobacter sphaeroides, ∆p was determined under anaerobic conditions in the dark, and the ATPase activity has been studied in R. sphaeroides strain A-10, isolated from Arzni mineral springs in Armenia. Membrane potential (∆φ) was measured from the distribution of tetraphenylphosphonium cation; pH gradient (∆pH) was the difference between the external and cytoplasmic pH values, and the latter was measured by 9-aminoacridine (9-AA) fluorescence changes. At pH 7.5, ∆φ was of −94 mV and the reversed ∆pH was +30 mV, resulting in ∆p of −64 mV. The addition of N,N′-dicyclohexylcarbodiimide (DCCD), the F₀F₁–ATPase inhibitor, was not affect ∆φ. It was shown that ∆φ varies nearly linearly with ΔpH, ∆φ increased from −57.1 mV at pH 6.0 to −103.8 mV at pH 8.0; it was compensated at high external pH by a reversed ∆pH, resulting in a low ∆p under anaerobic-dark conditions. Intracellular ATP concentrations and energetic charge (EC) were measured to evaluate a metabolism activity of R. sphaeroides.     

Redundant roles of Srs2 helicase and replication checkpoint in survival and rDNA maintenance in Schizosaccharomyces pombe

Srs2 helicase is believed to function as an anti-recombinase by resolving inappropriate Rad51-DNA filament. We found synthetic lethality or poor growth of srs2 with rad3 or mrc1 in Schizossacharomyces pombe. Lethality may result from a defect in non-checkpoint function of Rad3 or Mrc1 in the absence of Srs2, because srs2∆ rad9∆, srs2∆ chk1∆ cds1∆ or srs2∆ mrc1-14A (non-phosphorylatable mrc1 allele) did not show significant growth impairment. Notably, the inactivation of rhp51/RAD51 or rad22/RAD52 failed to rescue the growth, suggesting that events that impose lethality are independent of homologous recombination. Incubation of the conditional srs2∆ rad3 ^ts cells at restrictive temperature led not only to a viability decrease but also to a remarkable shortening of rDNA clusters (~100 copies). As opposed to the growth defect, shortening of rDNA clusters was also observed in srs2∆ rad9∆, srs2∆ chk1∆ cds1∆ or srs2∆ mrc1-14A, indicating that proper replication checkpoint signaling is critical for rDNA maintenance. Activation of Chk1 in the unchallenged mrc1-14A srs2∆ cells implies a certain level of spontaneous fork damage that might be the cause for rDNA instability. The data suggest that redundant functions of Srs2 and checkpoint proteins are essential for two independent aspects of genome maintenance. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

MRE11 and COM1/SAE2 are required for double-strand break repair and efficient chromosome pairing during meiosis of the protist Tetrahymena

Programmed DNA double-strand breaks (DSBs) are generated during meiosis to initiate homologous recombination. Various aspects of DSB formation, signaling, and repair are accomplished or governed by Mre11, a component of the MRN/MRX complex, partially in cooperation with Com1/Sae2/CtIP. We used Tetrahymena to study evolutionarily conserved and changed functions of Mre11 and Com1. There is a difference between organisms with respect to the dependency of meiotic DSB formation on Mre11. By cytology and an electrophoresis-based assay for DSBs, we found that in Tetrahymena Mre11p is not required for the formation and ATR-dependent signaling of DSBs. Its dispensability is also reflected by wild-type-like DSB-dependent reorganization of the meiotic nucleus and by the phosphorylation of H2A.X in mre11∆ mutant. However, mre11∆ and com1∆ mutants are unable to repair DSBs, and chromosome pairing is reduced. It is concluded that, while MRE11 has no universal role in DNA damage signaling, its requirement for DSB repair is conserved between evolutionarily distant organisms. Moreover, reduced chromosome pairing in repair-deficient mutants reveals the existence of two complementing pairing processes, one by the rough parallel arrangement of chromosomes imposed by the tubular shape of the meiotic nucleus and the other by repair-dependent precise sequence matching.     

ChMCO1 of Cochliobolus heterostrophus is a new class of metallo-oxidase, playing an important role in DHN-melanization

A metallo-oxidase gene from a phytopathogenic filamentous fungus, Cochliobolus heterostrophus was cloned. Structural prediction of ChMco1 indicated that this protein lacks a transmembrane helix and is soluble, whereas other known fungal metallo-oxidases including Saccharomyces cerevisiae FET3 are localized to the cell membrane. The results of searches in fungal genomic databases and phylogenetic analysis of fungal metallo-oxidases revealed that ChMco1 and its allies are distinct homologues of Fet3 and unique to filamentous ascomycetous species including C. heterostrophus. We performed a functional analysis of ChMCO1 by generating null mutants for the ChMco1 gene. The ChMco1 null (∆ChMco1) mutants clearly had reduced melanization, although they showed normal growth and conidiation. Results also show that ∆ChMco1 mutants lost laccase activity. These results suggest that ChMCO1 is a novel class of metallo-oxidase that is necessary for laccase activity and melanization.     

The FRP1 F-box gene has different functions in sexuality, pathogenicity and metabolism in three fungal pathogens

Plant‐pathogenic fungi employ a variety of infection strategies; as a result, fungi probably rely on different sets of proteins for successful infection. The F‐box protein Frp1, only present in filamentous fungi belonging to the Sordariomycetes, Leotiomycetes and Dothideomycetes, is required for nonsugar carbon catabolism and pathogenicity in the root‐infecting fungus Fusarium oxysporum. To assess the role of Frp1 in other plant‐pathogenic fungi, FRP1 deletion mutants were generated in Fusarium graminearum and Botrytis cinerea, and their phenotypes were analysed. Deletion of FgFRP1 in F. graminearum led to impaired infection of barley roots, but not of aerial plant parts. Deletion of BcFRP1 in B. cinerea did not show any effect on pathogenicity. Sexual reproduction, however, was impaired in both F. graminearum and B. cinerea FRP1 deletion mutants. The mutants of all three fungi displayed different phenotypes when grown on an array of carbon sources. The F. oxysporum and B. cinerea deletion mutants showed opposite growth phenotypes on sugar and nonsugar carbon sources. Replacement of FoFRP1 in F. oxysporum with the B. cinerea BcFRP1 resulted in the restoration of pathogenicity, but also in a switch from impaired growth on nonsugar carbon sources to impaired growth on sugar carbon sources. This effect could be ascribed in part to the B. cinerea BcFRP1 promoter sequence. In conclusion, the function of the F‐box protein Frp1, despite its high sequence conservation, is not conserved between different fungi, leading to differential requirements for pathogenicity and carbon source utilization.     

A proteomic study of pectin‐degrading enzymes secreted by Botrytis cinerea grown in liquid culture

Botrytis cinerea is a pathogenic filamentous fungus, which infects more than 200 plant species. The enzymes secreted by B. cinerea play an important role in the successful colonization of a host plant. Some of the secreted enzymes are involved in the degradation of pectin, a major component of the plant cell wall. A total of 126 proteins secreted by B. cinerea were identified by growing the fungus on highly or partially esterified pectin, or on sucrose in liquid culture. Sixty‐seven common proteins were identified in each of the growth conditions, of which 50 proteins exhibited a SignalP motif. Thirteen B. cinerea proteins with functions related to pectin degradation were identified in both pectin growth conditions, while only four were identified in sucrose. Our results indicate it is unlikely that the activation of B. cinerea from the dormant state to active infection is solely dependent on changes in the degree of esterification of the pectin component of the plant cell wall. Further, these results suggest that future studies of the B. cinerea secretome in infections of ripe and unripe fruits will provide important information that will describe the mechanisms that the fungus employs to access nutrients and decompose tissues.     

Functional analysis of an extracellular catalase of Botrytis cinerea

There is evidence that the necrotrophic fungal pathogen Botrytis cinerea is exposed to oxidative processes within plant tissues. The pathogen itself also generates active oxygen species and H₂O₂ as pathogenicity factors. Our aim was to study how the pathogen may defend itself against cellular damage caused by the accumulation of H₂O₂ and the role of an extracellular catalase in its detoxification during the infection of tomato and bean plants by B. cinerea. Chloronaphthol staining followed by light microscopy showed that H₂O₂ accumulates in the infection zone in tomato and bean leaves. An extracellular catalase gene (denominated Bccat2) was cloned from B. cinerea. Exposure of mycelium to H₂O₂ in liquid culture resulted in increased Bccat2 mRNA levels in a concentration-dependent manner. Bccat2 mRNA was detected at early stages of tomato leaf infection, suggesting that B. cinerea experiences oxidative stress. Bccat2-deficient mutants were generated by transformation-mediated gene disruption. Mutants were more sensitive then the wild-type strain to H₂O₂in vitro, but they partly compensated for the absence of BcCAT2 by activating other protective mechanisms in the presence of H₂O₂. Bccat2-deficient mutants did not display a consistent reduction of virulence on bean and tomato leaves. Cerium chloride staining of infected leaf tissue for ultrastructural studies showed that Bccat2-deficient mutants were exposed to H₂O₂ comparably to the wild-type. The results suggest that B. cinerea is a robust pathogen adapted to growing in hostile oxidizing environments in host tissues.     

Proteomic analysis of the phytopathogenic fungus Botrytis cinerea during cellulose degradation

The ascomycete Botrytis cinerea is a phytopathogenic fungus infecting and causing significant yield losses in a number of crops. Moreover, in the last few years, B. cinerea has been adopted as an important model system in molecular phytopathology. In spite of these contributions, the molecular basis of the infection cycle remains unclear. Proteomic approaches have revealed significant information about the infective cycle of several pathogens, including B. cinerea. The main aim of this study is to make available a proteomic database containing a significant number of identified proteins from B. cinerea. In brief, three independent B. cinerea cultures supplemented with carboxymethylcellulose were used, and the extracted proteins were independently separated by 2‐D PAGE to obtain the proteome map from B. cinerea. Two hundred and sixty‐seven spots were selected for MALDI TOF/TOF MS analysis, resulting in 303 positive identifications, mostly representing unannotated proteins. Identified proteins were then classified into categories using the PANTHER classification system ( www.pantherdb.org ), showing the relevance of protein metabolism and modification process and oxidoreductase activity. Since cellulose is one of the major components of the plant cell wall, many of the identified proteins may have a crucial role in the pathogenicity process. In brief, this proteomic map of B. cinerea will be a useful basis for exploring the proteins involved in the infection cycle, which will in turn provide new targets for crop diagnosis and focused fungicide design.     

Characterization of Botrytis–plant interactions using PathTrack©—an automated system for dynamic analysis of disease development

The measurement of disease development is integral in studies on plant–microbe interactions. To address the need for a dynamic and quantitative disease evaluation, we developed PathTrack^©, and used it to analyse the interaction of plants with Botrytis cinerea. PathTrack^© is composed of an infection chamber, a photography unit and software that produces video files and numerical values of disease progression. We identified a previously unrecognized infection stage and determined numerical parameters of pathogenic development. Using these parameters, we identified differences in disease dynamics between seemingly similar B. cinerea pathogenicity mutants, and revealed new details on plant susceptibility to the fungus. We showed that the difference between the lesion expansion rate on leaves and colony spreading rate on artificial medium reflects the levels of the plant immune system, suggesting that this parameter can be used to quantify plant defence. Our results shed new light and reveal new details of the interaction between the model necrotrophic pathogen B. cinerea and plants. The concept that we present is universal and may be applied to facilitate the study of various types of plant–pathogen association.     

The ABC-transporter AtmA is involved in nickel and cobalt resistance of <Emphasis Type="Italic">Cupriavidus metallidurans</Emphasis> strain CH34

Cupriavidus metallidurans CH34 genome contains an ortholog of Atm1p named AtmA (Rmet_0391, YP_582546). In Saccharomyces cerevisiae, the ABC-type transport system Atm1p is involved in export of iron–sulfur clusters from mitochondria into the cytoplasm for assembly of cytoplasmic iron–sulfur containing proteins. An ∆atmA mutant of C. metallidurans was sensitive to nickel and cobalt but not iron cations. AtmA increased also resistance to these cations in Escherichia coli strains that carry deletions of the genes for other nickel and cobalt transport systems. In C. metallidurans, atmA expression was not significantly induced by nickel and cobalt, but repressed by zinc. AtmA was purified as a 70 kDa protein after expression in E. coli. ATPase activity of AtmA was stimulated by nickel and cobalt.     

Phylogenetic analysis of heavy-metal ATPases in fungi and characterization of the copper-transporting ATPase of Cochliobolus heterostrophus

We performed a phylogenetic analysis of heavy-metal ATPases (HMAs) in fungi and found that HMAs can be divided into three groups, A, B, and C. Group A is predicted to deliver copper ions to copper-containing proteins, while Groups B and C are thought to function as cell-membrane copper-efflux pumps. Furthermore, Groups B and C consist of fungal-specific HMAs, while Group A consists of fungal orthologues that have been well conserved in eukaryotes. We also cloned and characterized a Group A-type HMA gene (i.e., ChCcc2) of the filamentous plant pathogen, Cochliobolus heterostrophus. Mutation of ChCcc2 severely affected growth, pigmentation, conidiation, and colonial morphology. Activity of the copper-containing protein, laccase, was also lost in ChCcc2 mutants, suggesting that ChCCC2 plays an important role in growth and morphology by activating various copper-containing proteins in C. heterostrophus.     

The infection cushion of Botrytis cinerea: a fungal ‘weapon’ of plant-biomass destruction

The necrotrophic plant-pathogen fungus Botrytis cinerea produces multicellular appressoria dedicated to plant penetration, named infection cushions (IC). A microarray analysis was performed to identify genes upregulated in mature IC. The expression data were validated by RT-qPCR analysis performed in vitro and in planta, proteomic analysis of the IC secretome and biochemical assays. 1231 upregulated genes and 79 up-accumulated proteins were identified. The data support the secretion of effectors by IC: phytotoxins, ROS, proteases, cutinases, plant cell wall–degrading enzymes and plant cell death–inducing proteins. Parallel upregulation of sugar transport and sugar catabolism–encoding genes would indicate a role of IC in nutrition. The data also reveal a substantial remodelling of the IC cell wall and suggest a role for melanin and chitosan in IC function. Lastly, mutagenesis of two upregulated genes in IC identified secreted fasciclin-like proteins as actors in the pathogenesis of B. cinerea. These results support the role of IC in plant penetration and also introduce other unexpected functions for this fungal organ, in colonization, necrotrophy and nutrition of the pathogen.     

Overexpression of a Monocot Acyl‐CoA‐Binding Protein Confers Broad‐Spectrum Pathogen Protection in a Dicot

Plants are continuously infected by various pathogens throughout their lifecycle. Previous studies have reported that the expression of Class III acyl‐CoA‐binding proteins (ACBPs) such as the Arabidopsis ACBP3 and rice ACBP5 were induced by pathogen infection. Transgenic Arabidopsis AtACBP3‐overexpressors (AtACBP3‐OEs) displayed enhanced protection against the bacterial biotroph, Pseudomonas syringae, although they became susceptible to the fungal necrotroph Botrytis cinerea. A Class III ACBP from a monocot, rice (Oryza sativa) OsACBP5 was overexpressed in the dicot Arabidopsis. The resultant transgenic Arabidopsis lines conferred resistance not only to the bacterial biotroph P. syringae but to fungal necrotrophs (Rhizoctonia solani, B. cinerea, Alternaria brassicicola) and a hemibiotroph (Colletotrichum siamense). Changes in protein expression in R. solani‐infected Arabidopsis OsACBP5‐overexpressors (OsACBP5‐OEs) were demonstrated using proteomic analysis. Biotic stress‐related proteins including cell wall‐related proteins such as FASCILIN‐LIKE ARABINOGALACTAN‐PROTEIN10, LEUCINE‐RICH REPEAT EXTENSIN‐LIKE PROTEINS, XYLOGLUCAN ENDOTRANSGLUCOSYLASE/HYDROLASE PROTEIN4, and PECTINESTERASE INHIBITOR18; proteins associated with glucosinolate degradation including GDSL‐LIKE LIPASE23, EPITHIOSPECIFIER MODIFIER1, MYROSINASE1, MYROSINASE2, and NITRILASE1; as well as a protein involved in jasmonate biosynthesis, ALLENE OXIDE CYCLASE2, were induced in OsACBP5‐OEs upon R. solani infection. These results indicated that upregulation of these proteins in OsACBP5‐OEs conferred protection against various plant pathogens.