Sequence variation in the CYP51 gene of Blumeria graminis associated with resistance to sterol demethylase inhibiting fungicides

Resistance to sterol 14alpha-demethylase inhibiting fungicides (DMIs) has been correlated with mutations in the CYP51 gene, which encodes the target enzyme eburicol 14alpha-demethylase. To test the hypothesis that variation in the CYP51 gene explains variation for DMI sensitivity in barley and wheat powdery mildew species, this gene was sequenced from isolates of Blumeria graminis f.sp. hordei (Bgh) and f.sp. tritici (Bgt), respectively, which differed in their responses to DMIs in agricultural populations in the UK. Two single-nucleotide mutations in the CYP51 gene, which resulted in the amino acid substitutions Y136F and K147Q, were detected. K147Q is a novel mutation present only in Bgh isolates expressing very high levels of resistance. Sequence analysis of the CYP51 gene from the progeny of a cross between DMI-sensitive and resistant Bgh isolates showed that both mutations segregate with resistance, which is consistent with CYP51 controlling a major portion of DMI resistance. However, genetic analysis of resistance to the DMI triadimenol indicates that mutation of the CYP51 gene is not the only mechanism of resistance operating in B. graminis.     

Transient transformation of the rust fungus Puccinia graminis f. sp. tritici

The biotrophic rust fungus Puccinia graminis f. sp. tritici (Pgt) was transformed by particle bombardment. The promoter from the Pgt translation elongation factor 1alpha (EF-1alpha) gene was fused to the bacterial marker genes hygromycin B phosphotransferase (hpt) and beta-glucuronidase (GUS). Transformation constructs were introduced into uredospores of Pgt, an obligate pathogen of wheat, by biolistic bombardment. Uredospores transformed with the construct containing the hpt gene germinated and initiated branching on selective medium, indicating that they had acquired resistance to hygromycin B. However, transformants stopped growing 5 days after bombardment. GUS activity in uredospores and germlings was histochemically detected 4-16 h after bombardment. GUS expression was also obtained using the INF24 promoter from the bean rust fungus Uromyces appendiculatus, demonstrating that heterologous genes can be expressed in P. graminis under the control of regulatory sequences from closely related organisms.     

Anti-apoptotic machinery protects the necrotrophic fungus Botrytis cinerea from host-induced apoptotic-like cell death during plant infection

Necrotrophic fungi are unable to occupy living plant cells. How such pathogens survive first contact with living host tissue and initiate infection is therefore unclear. Here, we show that the necrotrophic grey mold fungus Botrytis cinerea undergoes massive apoptotic-like programmed cell death (PCD) following germination on the host plant. Manipulation of an anti-apoptotic gene BcBIR1 modified fungal response to PCD-inducing conditions. As a consequence, strains with reduced sensitivity to PCD were hyper virulent, while strains in which PCD was over-stimulated showed reduced pathogenicity. Similarly, reduced levels of PCD in the fungus were recorded following infection of Arabidopsis mutants that show enhanced susceptibility to B. cinerea. When considered together, these results suggest that Botrytis PCD machinery is targeted by plant defense molecules, and that the fungal anti-apoptotic machinery is essential for overcoming this host-induced PCD and hence, for establishment of infection. As such, fungal PCD machinery represents a novel target for fungicides and antifungal drugs.     

Molecular and functional characterization of a fructose specific transporter from the gray mold fungus Botrytis cinerea

In the gray mold fungus Botrytis cinerea, spore germination and plant infection are stimulated in the presence of nutrients, in particular sugars. Applied at micromolar concentrations, fructose is a more potent inducer of germination than glucose. To test whether preferred fructose uptake is responsible for this effect, and to study the mechanism of fructose transport in B. cinerea, a gene (frt1) encoding a fructose transporter was cloned. FRT1 is highly similar to recently identified fructose transporters of yeasts, but much less to other fungal hexose transporters characterized so far. By using a hexose uptake deficient yeast strain for expression, FRT1 was found to be a high affinity proton coupled symporter specific for fructose but not for glucose. B. cinerea frt1 disruption mutants were created and showed normal vegetative growth and plant infection, but a delay in fructose-induced germination when compared to wild-type. Sugar uptake experiments with both wild-type and mutant conidia showed a higher affinity for glucose than for fructose. Thus, we propose that the specific effect of fructose on germination is not due to transport but rather to an as yet unknown intracellular sensing.     

Agrobacterium tumefaciens-mediated genetic transformation of the entomopathogenic fungus Beauveria bassiana

Agrobacterium tumefaciens-mediated transformation (agro-transformation) was successfully applied to the entomogenous fungus Beauveria bassiana. Conidia of B. bassiana were transformed to hygromycin B resistance using the hph gene of Escherichia coli as the selective trait, under the control of a heterologous fungal promoter and the Aspergillus nidulans trpC terminator. The efficiency of transformation was up to 28 and 96 transformants per 10(4) and 10(5) target conidia, respectively, using three distinct vectors. High mitotic stability of the transformants (80-100%) was demonstrated after five successive transfers on a nonselective medium. Abortive transformants were observed for all the hph(r) vectors used. Putative transformants were analysed for the presence of the hph gene by PCR and Southern analysis. The latter analysis revealed the integration of two or more copies of the hph gene in the genome. The agro-transformation method was found to be effective for the isolation of B. bassiana hygromycin resistant transformants and may represent a useful tool for insertional mutagenesis studies in this fungus.     

A sterol 14α-demethylase is required for conidiation, virulence and for mediating sensitivity to sterol demethylation inhibitors by the rice blast fungus Magnaporthe oryzae

The Magnaporthe oryzae genome contains two homologous CYP51 genes, MoCYP51A and MoCYP51B, that putatively encode sterol 14α-demethylase enzymes. Targeted gene deletion mutants of MoCYP51A were morphologically indistinguishable from the isogenic wild type M. oryzae strain Guy11 in vegetative culture, but were impaired in both conidiation and virulence. Deletion of MoCYP51B did not result in any obvious phenotypic changes compared with Guy11. The Δmocyp51A mutants were also highly sensitive to sterol demethylation inhibitor (DMI) fungicides, while Δmocyp51B mutants were unchanged in their sensitivity to these fungicides. Expression of both MoCYP51A and MoCYP51B was significantly induced by exposure to DMI fungicides. Analysis of intracellular localization of MoCyp51A showed that MoCyp51A was mainly localized to the cytoplasm of hyphae and conidia. Taken together, our results indicate that MoCYP51A is required for efficient conidiogenesis, full virulence and for mediating DMI sensitivity by the rice blast fungus.     

Genetic transformation system for the aflatoxin-producing fungus Aspergillus flavus

A heterologous transformation system was developed for Aspergillus flavus with efficiencies greater than 20 stable transformants per micrograms of DNA. Protoplasts of uracil-requiring strains of the fungus were transformed with plasmid and cosmid vectors containing the pyr-4 gene of Neurospora crassa. Transformants were selected for their ability to grow and sporulate on medium lacking uracil. Vector DNA appeared to integrate randomly into the genome of A. flavus with a tendency for multiple, tandem insertion. Transformants with single or multiple insertions were stable after five consecutive transfers on medium containing uracil. Uracil-requiring recipient strains were obtained either by UV-irradiating conidia and selecting colonies resistant to 5-fluoroorotic acid or by transferring the mutated pyr locus to strains by parasexual recombination. This is the first report of a transformation system for an aflatoxin-producing fungus. The transformation system and the availability of aflatoxin-negative mutants provide a new approach to studying the biosynthesis and regulation of aflatoxin.     

Plant signalling components EDS1 and SGT1 enhance disease caused by the necrotrophic pathogen Botrytis cinerea

* Botrytis cinerea is a necrotrophic fungus that causes grey mould on a wide range of food plants, especially grapevine, tomato, soft fruits and vegetables. This disease brings about important economic losses in both pre- and postharvest crops. Successful protection of host plants against this pathogen is severely hampered by a lack of resistance genes in the hosts and the considerable phenotypic diversity of the fungus. * The aim of this study was to test whether B. cinerea manipulates the immunity-signalling pathways in plants to restore its disease. * We showed that B. cinerea caused disease in Nicotiana benthamiana through the activation of two plant signalling genes, EDS1 and SGT1, which have been shown to be essential for resistance against biotrophic pathogens; and more interestingly, virus-induced gene silencing of these two plant signalling components enhanced N. benthamiana resistance to B. cinerea. Finally, plants expressing the baculovirus antiapoptotic protein p35 were more resistant to this necrotrophic pathogen than wild-type plants. * This work highlights a new strategy used by B. cinerea to establish disease. This information is important for the design of strategies to improve plant pathogen resistance.     

Genetic transformation system for the aflatoxin-producing fungus Aspergillus flavus.

A heterologous transformation system was developed for Aspergillus flavus with efficiencies greater than 20 stable transformants per micrograms of DNA. Protoplasts of uracil-requiring strains of the fungus were transformed with plasmid and cosmid vectors containing the pyr-4 gene of Neurospora crassa. Transformants were selected for their ability to grow and sporulate on medium lacking uracil. Vector DNA appeared to integrate randomly into the genome of A. flavus with a tendency for multiple, tandem insertion. Transformants with single or multiple insertions were stable after five consecutive transfers on medium containing uracil. Uracil-requiring recipient strains were obtained either by UV-irradiating conidia and selecting colonies resistant to 5-fluoroorotic acid or by transferring the mutated pyr locus to strains by parasexual recombination. This is the first report of a transformation system for an aflatoxin-producing fungus. The transformation system and the availability of aflatoxin-negative mutants provide a new approach to studying the biosynthesis and regulation of aflatoxin.     

Cloning and partial characterization of endopolygalacturonase genes from Botrytis cinerea

Botrytis cinerea is a plant-pathogenic fungus infecting over 200 different plant species. We use a molecular genetic approach to study the process of pectin degradation by the fungus. Recently, we described the cloning and characterization of an endopolygalacturonase (endoPG) gene from B. cinerea (Bcpg1) which is required for full virulence. Here we describe the cloning and characterization of five additional endoPG-encoding genes from B. cinerea SAS56. The identity at the amino acid level between the six endoPGs of B. cinerea varied from 34 to 73%. Phylogenetic analysis, by using a group of 35 related fungal endoPGs and as an outgroup one plant PG, resulted in the identification of five monophyletic groups of closely related proteins. The endoPG proteins from B. cinerea SAS56 could be assigned to three different monophyletic groups. DNA blot analysis revealed the presence of the complete endoPG gene family in other strains of B. cinerea, as well as in other Botrytis species. Differential gene expression of the gene family members was found in mycelium grown in liquid culture with either glucose or polygalacturonic acid as the carbon source.     

beta-Aminobutyric acid-induced protection of Arabidopsis against the necrotrophic fungus Botrytis cinerea

The non-protein amino acid beta-aminobutyric acid (BABA) protects numerous plants against various pathogens. Protection of Arabidopsis plants against virulent pathogens involves the potentiation of pathogen-specific defense responses. To extend the analysis of the mode of action of BABA to necrotrophs we evaluated the effect of this chemical on Arabidopsis plants infected with the gray mold fungus Botrytis cinerea. BABA-treated Arabidopsis were found to be less sensitive to two different strains of this pathogen. BABA protected mutants defective in the jasmonate and ethylene pathways, but was inactive in plants impaired in the systemic acquired resistance transduction pathway. Treatments with benzo-(1,2,3)-thiadiazole-7-carbothioic acid S-methyl ester, a functional analog of salicylic acid (SA), also markedly reduced the level of infection. Moreover, BABA potentiated mRNA accumulation of the SA-associated PR-1, but not the jasmonate/ethylene-dependent PDF1.2 gene. Thus, besides jasmonate/ethylene-dependent defense responses, SA-dependent signaling also contributes to restrict B. cinerea infection in Arabidopsis. Our results also suggest that SA-dependent signaling is down-regulated after infection by B. cinerea. The observed up-regulation of the PDF1.2 gene in mutants defective in the SA-dependent signaling pathway points to a cross-talk between SA- and jasmonate/ethylene-dependent signaling pathways during pathogen ingress.     

Transformation of the nematode-trapping fungus Arthrobotrys oligospora

The nematode-trapping fungus Arthrobotrys oligospora was transformed to hygromycin resistance using the hygromycin-B phosphotransferase gene from Escherichia coli under the control of various heterologous fungal promoters. Plasmid DNA was introduced into fungal protoplasts by polyethylene glycol/CaCl2 treatment. Transformation frequencies varied between 1-6 transformants per microgram DNA. Seven out of 13 integration events analyzed from transformants were single copy integrations, whereas the remaining were multiple and more complex integrations. The addition of restriction enzymes during transformations increased the frequency of single copy integrations. Co-transformation, using the E. coli uidA gene encoding the beta-glucuronidase reporter gene under the control of an Aspergillus nidulans promoter, occurred at frequencies of up to 63%.     

Living colors in the gray mold pathogen Botrytis cinerea: codon-optimized genes encoding green fluorescent protein and mCherry, which exhibit bright fluorescence

The green fluorescent protein (GFP) and its variants have been widely used in modern biology as reporters that allow a variety of live-cell imaging techniques. So far, GFP has rarely been used in the gray mold fungus Botrytis cinerea because of low fluorescence intensity. The codon usage of B. cinerea genes strongly deviates from that of commonly used GFP-encoding genes and reveals a lower GC content than other fungi. In this study, we report the development and use of a codon-optimized version of the B. cinerea enhanced GFP (eGFP)-encoding gene (Bcgfp) for improved expression in B. cinerea. Both the codon optimization and, to a smaller extent, the insertion of an intron resulted in higher mRNA levels and increased fluorescence. Bcgfp was used for localization of nuclei in germinating spores and for visualizing host penetration. We further demonstrate the use of promoter-Bcgfp fusions for quantitative evaluation of various toxic compounds as inducers of the atrB gene encoding an ABC-type drug efflux transporter of B. cinerea. In addition, a codon-optimized mCherry-encoding gene was constructed which yielded bright red fluorescence in B. cinerea.     

The BMP1 gene is essential for pathogenicity in the gray mold fungus Botrytis cinerea

In Magnaporthe grisea, a well-conserved mitogen-activated protein (MAP) kinase gene, PMK1, is essential for fungal pathogenesis. In this study, we tested whether the same MAP kinase is essential for plant infection in the gray mold fungus Botrytis cinerea, a necrotrophic pathogen that employs infection mechanisms different from those of M. grisea. We used a polymerase chain reaction-based approach to isolate MAP kinase homologues from B. cinerea. The Botrytis MAP kinase required for pathogenesis (BMP) MAP kinase gene is highly homologous to the M. grisea PMK1. BMP1 is a single-copy gene. bmp1 gene replacement mutants produced normal conidia and mycelia but were reduced in growth rate on nutrient-rich medium. bmp1 mutants were nonpathogenic on carnation flowers and tomato leaves. Re-introduction of the wild-type BMP1 allele into the bmp1 mutant restored both normal growth rate and pathogenicity. Further studies indicated that conidia from bmp1 mutants germinated on plant surfaces but failed to penetrate and macerate plant tissues. bmp1 mutants also appeared to be defective in infecting through wounds. These results indicated that BMP1 is essential for plant infection in B. cinerea, and this MAP kinase pathway may be widely conserved in pathogenic fungi for regulating infection processes.