Evolution of the EKA family of powdery mildew avirulence-effector genes from the ORF 1 of a LINE retrotransposon.

BACKGROUND: The Avrk1 and Avra10 avirulence (AVR) genes encode effectors that increase the pathogenicity of the fungus Blumeria graminis f.sp. hordei (Bgh), the powdery mildew pathogen, in susceptible barley plants. In resistant barley, MLK1 and MLA10 resistance proteins recognize the presence of AVRK1 and AVRA10, eliciting the hypersensitive response typical of gene for gene interactions. Avrk1 and Avra10 have more than 1350 homologues in Bgh genome, forming the EKA (Effectors homologous to Avr k 1 and Avr a 10) gene family. RESULTS: We tested the hypothesis that the EKA family originated from degenerate copies of Class I LINE retrotransposons by analysing the EKA family in the genome of Bgh isolate DH14 with bioinformatic tools specially developed for the analysis of Transposable Elements (TE) in genomes. The Class I LINE retrotransposon copies homologous to Avrk1 and Avra10 represent 6.5 % of the Bgh annotated genome and, among them, we identified 293 AVR/effector candidate genes. We also experimentally identified peptides that indicated the translation of several predicted proteins from EKA family members, which had higher relative abundance in haustoria than in hyphae. CONCLUSIONS: Our analyses indicate that Avrk1 and Avra10 have evolved from part of the ORF1 gene of Class I LINE retrotransposons. The co-option of Avra10 and Avrk1 as effectors from truncated copies of retrotransposons explains the huge number of homologues in Bgh genome that could act as dynamic reservoirs from which new effector genes may evolve. These data provide further evidence for recruitment of retrotransposons in the evolution of new biological functions.

Designer laccases: a vogue for high-potential fungal enzymes?

Laccases are blue multicopper oxidases that catalyse the four-electron reduction of O(2) to water coupled with the oxidation of small organic substrates. Secreted basidiomycete white-rot fungal laccases orchestrate this with high thermodynamic efficiency, making these enzymes excellent candidates for exploitation as industrial oxidants. However, these fungi are less tractable genetically than the ascomycetes, which predominantly produce lower-potential laccases. We address the state-of-play regarding expression of high reduction potential laccases in heterologous hosts, and issues regarding enzyme glycosylation status. We describe the synergistic role of structural biology, particularly in unmasking structure-function relationships following genetic modification and their collective impact on laccase yields. Such recent research draws closer the prospect of industrial quantities of designer, fit-for-purpose laccases.

Coevolution between a family of parasite virulence effectors and a class of LINE-1 retrotransposons.

Parasites are able to evolve rapidly and overcome host defense mechanisms, but the molecular basis of this adaptation is poorly understood. Powdery mildew fungi (Erysiphales, Ascomycota) are obligate biotrophic parasites infecting nearly 10,000 plant genera. They obtain their nutrients from host plants through specialized feeding structures known as haustoria. We previously identified the AVR(k1) powdery mildew-specific gene family encoding effectors that contribute to the successful establishment of haustoria. Here, we report the extensive proliferation of the AVR(k1) gene family throughout the genome of B. graminis, with sequences diverging in formae speciales adapted to infect different hosts. Also, importantly, we have discovered that the effectors have coevolved with a particular family of LINE-1 retrotransposons, named TE1a. The coevolution of these two entities indicates a mutual benefit to the association, which could ultimately contribute to parasite adaptation and success. We propose that the association would benefit 1) the powdery mildew fungus, by providing a mechanism for amplifying and diversifying effectors and 2) the associated retrotransposons, by providing a basis for their maintenance through selection in the fungal genome.

Against the grain: safeguarding rice from rice blast disease.

Rice is the staple diet of more than three billion people. Yields must double over the next 40 years if we are to sustain the nutritional needs of the ever-expanding global population. Between 10% and 30% of the annual rice harvest is lost due to infection by the rice blast fungus Magnaporthe oryzae. Evaluation of genetic and virulence diversity of blast populations with diagnostic markers will aid disease management. We review the M. oryzae species-specific and cultivar-specific avirulence determinants and evaluate efforts towards generating durable and broad-spectrum resistance in single resistant cultivars or mixtures. We consider modern usage of fungicides and plant defence activators, assess the usefulness of biological control and categorize current approaches towards blast-tolerant genetically modified rice.

Evolutionary history of the ancient cutinase family in five filamentous Ascomycetes reveals differential gene duplications and losses and in Magnaporthe grisea shows evidence of sub- and neo-functionalization.

* The cuticle is the first barrier for fungi that parasitize plants systematically or opportunistically. Here, the evolutionary history is reported of the multimembered cutinase families of the plant pathogenic Ascomycetes Magnaporthe grisea, Fusarium graminearum and Botrytis cinerea and the saprotrophic Ascomycetes Aspergillus nidulans and Neurospora crassa. * Molecular taxonomy of all fungal cutinases demonstrates a clear division into two ancient subfamilies. No evidence was found for lateral gene transfer from prokaryotes. The cutinases in the five Ascomycetes show significant copy number variation, they form six clades and their extreme sequence diversity is highlighted by the lack of consensus intron. The average ratio of gene duplication to loss is 2 : 3, with the exception of M. grisea and N. crassa, which exhibit extreme family expansion and contraction, respectively. * Detailed transcript profiling in vivo, categorizes the M. grisea cutinases into four regulatory patterns. Symmetric or asymmetric expression profiles of phylogenetically related cutinase genes suggest subfunctionalization and neofunctionalization, respectively. * The cutinase family-size per fungal species is discussed in relation to genome characteristics and lifestyle. The ancestry of the cutinase gene family, together with the expression divergence of its individual members provides a first insight into the drivers for niche differentiation in fungi.

Host perception and signal transduction studies in wild-type Blumeria graminis f. sp. hordei and a quinoxyfen-resistant mutant implicate quinoxyfen in the inhibition of serine esterase activity.

BACKGROUND: Quinoxyfen is a potent and effective fungicide, hitherto considered to control powdery mildew disease by perturbing signal transduction during early germling differentiation. The aim of this paper is to understand the mode of action of quinoxyfen by comparing the perception of host-derived signals and signal relay in a wild-type Blumeria graminis f. sp. hordei EM Marchal (Bgh) (WT/IM82) and a quinoxyfen-resistant field isolate (QR/2B11). RESULTS: QR/2B11 germinates more promiscuously on host-like and artificial surfaces than the quinoxyfen-sensitive WT/IM82. The pivotal role of host cuticle deprivation in the formation of hooked appressorial germ tubes (hAGTs) in WT/IM82 and a dramatic drop in germling differentiation in the presence of the mildewicide are demonstrated. QR/2B11 strain shows a dependence on host cuticle-like features for hAGT formation but no significant difference between germling differentiation in the presence or absence of quinoxyfen. PKC-inhibitor Ro 318220 induces morphological changes similar to those seen in quinoxyfen-treated germlings. PKC1 transcript accumulation is equivalently upregulated by quinoxyfen in QR/2B11 and WT/IM82 strains, but Bgh cutinase CUT1 transcript is 8 times more abundant in QR/2B11 conidia than in WT/IM82 conidia. Quinoxyfen inhibits serine esterase activity in WT/IM82, but not in QR/2B11. CONCLUSION: Collectively, these data suggest that quinoxyfen interferes with the perception of host-derived signals required for full germling differentiation, and that QR/2B11 bypasses the need for such signals. Moreover, quinoxyfen appears to target serine esterase activity, with a downstream perturbation in signal transduction; this represents the first demonstrable biochemical difference between the quinoxyfen-resistant and -sensitive isolates.

The fate of gene duplicates in the genomes of fungal pathogens.

Understanding how molecular changes underlie phenotypic variation within and between species is one of the main goals of evolutionary biology and comparative genetics. The recent proliferation of sequenced fungal genomes offers a unique opportunity to start elucidating the extreme phenotypic diversity in the Kingdom Fungi.1-4 We attempted to investigate the contribution of gene families to the evolutionary forces shaping the diversity of pathogenic lifestyles among the fungi.5 We studied a family of secreted enzymes which is present and expanded in all genomes of fungal pathogens sequenced to date and absent from the genomes of true yeasts.3,4 This family of cutinases6 predates the division between the two major fungal phyla, Ascomycota and Basidiomycota.5 We discuss our molecular phylogenetic analyses, the number and sequence diversity, and gene gains and losses of cutinase family members between five Ascomycetes: the phytopathogens Magnaporthe oryzae, Fusarium graminearum and Botrytis cinerea; and the model organisms Neurospora crassa and Aspergillus nidulans.5 The functional characterization of three members of the M. oryzae cutinase family,6-10 coupled with the regulatory subfunctionalization and neofunctionalization of most gene pairs5 provide the first justification for the retention of paralogs after duplication and for gene redundancy in the genomes of fungal pathogens.

Genetics of avirulence genes in Blumeria graminis f.sp. hordei and physical mapping of AVR(a22) and AVR(a12).

Powdery mildew fungi are parasites that cause disease on a wide range of important crops. Plant resistance (R) genes, which induce host defences against powdery mildews, encode proteins that recognise avirulence (AVR) molecules from the parasite in a gene-for-gene manner. To gain insight into how virulence evolves in Blumeria graminis f.sp. hordei, associations between segregating AVR genes were established. As a prerequisite to the isolation of AVR genes, two loci were selected for further analysis. AVR(a22) is located in a tightly linked cluster comprising AVR(a10) and AVR(k1) as well as up to five other AVR genes. The ratio between physical and genetic distance in the cluster ranged between 0.7 and 35 kB/cM. The AVR(a22) locus was delimited by the previously isolated gene AVR(a10) and two cleaved amplified polymorphic sequence (CAPS) markers, 19H12R and 74E9L. By contrast, AVR(a12) was not linked to other AVR genes in two crosses. Bulk segregant analysis of over 100,000 AFLP fragments yielded two markers, ETAMTG-285 and PAAMACT-473, mapping 10 and 2cM from AVR(a12), respectively, thus delimiting AVR(a12) on one side. All markers obtained for AVR(a12) mapped proximal to it, indicating that the gene is located at the end of a chromosome. Three more AVR(a10) paralogues were identified at the locus interspersed among genes for metabolic enzymes and abundant repetitive elements, especially those homologous to the CgT1 class of retrotransposons. The flanking and close markers obtained will facilitate the isolation of AVR(a22) and AVR(a12) and provide useful tools for studies of the evolution of powdery mildew fungi in agriculture and nature.

Cutinase and hydrophobin interplay: A herald for pathogenesis?

Surface-penetrating phytopathogenic fungi frequently form appressoria. These are specialised infection structures pivotal to fungal ingress into the host. Recently, we demonstrated that one member of a family of cutinases in Magnaporthe grisea is involved in surface sensing, mediating appressorium differentiation and penetration peg formation and hence facilitates host penetration. Cutinase2 serves as an upstream activator of cAMP/PKA and DAG/PKC signalling cascades and is essential for full virulence. Here, we speculate on the role of rice blast hydrophobins as surface interactors facilitating fungal cutinase activity.

Magnaporthe grisea cutinase2 mediates appressorium differentiation and host penetration and is required for full virulence.

The rice blast fungus Magnaporthe grisea infects its host by forming a specialized infection structure, the appressorium, on the plant leaf. The enormous turgor pressure generated within the appressorium drives the emerging penetration peg forcefully through the plant cuticle. Hitherto, the involvement of cutinase(s) in this process has remained unproven. We identified a specific M. grisea cutinase, CUT2, whose expression is dramatically upregulated during appressorium maturation and penetration. The cut2 mutant has reduced extracellular cutin-degrading and Ser esterase activity, when grown on cutin as the sole carbon source, compared with the wild-type strain. The cut2 mutant strain is severely less pathogenic than the wild type or complemented cut2/CUT2 strain on rice (Oryza sativa) and barley (Hordeum vulgare). It displays reduced conidiation and anomalous germling morphology, forming multiple elongated germ tubes and aberrant appressoria on inductive surfaces. We show that Cut2 mediates the formation of the penetration peg but does not play a role in spore or appressorium adhesion, or in appressorial turgor generation. Morphological and pathogenicity defects in the cut2 mutant are fully restored with exogenous application of synthetic cutin monomers, cAMP, 3-isobutyl-1-methylxanthine, and diacylglycerol (DAG). We propose that Cut2 is an upstream activator of cAMP/protein kinase A and DAG/protein kinase C signaling pathways that direct appressorium formation and infectious growth in M. grisea. Cut2 is therefore required for surface sensing leading to correct germling differentiation, penetration, and full virulence in this model fungus.

A novel role for catalase B in the maintenance of fungal cell-wall integrity during host invasion in the rice blast fungus Magnaporthe grisea.

Asexual spores of the rice blast fungus germinate to produce a specialized and melanized infection structure, the appressorium, which is pivotal to successful plant penetration. To investigate whether Magnaporthe grisea counteracts the toxic burst of H2O2 localized beneath the site of attempted invasion, we examined the temporal expression of five candidate antioxidant genes. Of these, the putatively secreted large subunit catalase CATB gene was 600-fold up-regulated in vivo, coincident with penetration, and moderately up-regulated in vitro, in response to exogenous H2O2. Targeted gene replacement of CATB led to compromised pathogen fitness; the catB mutant displayed paler pigmentation and accelerated hyphal growth but lower biomass, poorer sporulation, fragile conidia and appressoria, and impaired melanization. The catB mutant was severely less pathogenic than Guy 11 on barley and rice, and its infectivity was further reduced on exposure to H2O2. The wild-type phenotype was restored by the reintroduction of CATB into the catB mutant We found no evidence to support a role for CATB in detoxification of the host-derived H2O2 at the site of penetration. Instead, we demonstrated that CATB plays a part in strengthening the fungal wall, a role of particular importance during forceful entry into the host.

Gene microarray analysis using angular distribution decomposition.

Clustering techniques have been widely used in the analysis of microarray data to group genes with similar expression profiles. The similarity of expression profiles and hence the results of clustering greatly depend on how the data has been transformed. We present a method that uses the relative expression changes between pairs of conditions and an angular transformation to define the similarity of gene expression patterns. The pairwise comparisons of experimental conditions can be chosen to reflect the purpose of clustering allowing control the definition of similarity between genes. A variational Bayes mixture modeling approach is then used to find clusters within the transformed data. The purpose of microarray data analysis is often to locate groups genes showing particular patterns of expression change and within these groups to locate specific target genes that may warrant further experimental investigation. We show that the angular transformation maps data to a representation from which information, in terms of relative regulation changes, can be automatically mined. This information can be then be used to understand the "features" of expression change important to different clusters allowing potentially interesting clusters to be easily located. Finally, we show how the genes within a cluster can be visualized in terms of their expression pattern and intensity change, allowing potential target genes to be highlighted within the clusters of interest.

Multiple avirulence paralogues in cereal powdery mildew fungi may contribute to parasite fitness and defeat of plant resistance.

Powdery mildews, obligate biotrophic fungal parasites on a wide range of important crops, can be controlled by plant resistance (R) genes, but these are rapidly overcome by parasite mutants evading recognition. It is unknown how this rapid evolution occurs without apparent loss of parasite fitness. R proteins recognize avirulence (AVR) molecules from parasites in a gene-for-gene manner and trigger defense responses. We identify AVR(a10) and AVR(k1) of barley powdery mildew fungus, Blumeria graminis f sp hordei (Bgh), and show that they induce both cell death and inaccessibility when transiently expressed in Mla10 and Mlk1 barley (Hordeum vulgare) varieties, respectively. In contrast with other reported fungal AVR genes, AVR(a10) and AVR(k1) encode proteins that lack secretion signal peptides and enhance infection success on susceptible host plant cells. AVR(a10) and AVR(k1) belong to a large family with >30 paralogues in the genome of Bgh, and homologous sequences are present in other formae speciales of the fungus infecting other grasses. Our findings imply that the mildew fungus has a repertoire of AVR genes, which may function as effectors and contribute to parasite virulence. Multiple copies of related but distinct AVR effector paralogues might enable populations of Bgh to rapidly overcome host R genes while maintaining virulence.