Pari Skamnioti

Genome Expansion and Gene Loss in Powdery Mildew Fungi Reveal Tradeoffs in Extreme Parasitism

From Blight to Powdery Mildew Pathogenic effects of microbes on plants have widespread consequences. Witness, for example, the cultural upheavals driven by potato blight in the 1800s. A variety of microbial pathogens continue to afflict crop plants today, driving both loss of yield and incurring the increased costs of control mechanisms. Now, four reports analyze microbial genomes in order to understand better how plant pathogens function (see the Perspective by Dodds). Raffaele et al. (p. 1540) describe how the genome of the potato blight pathogen accommodates transfer to different hosts. Spanu et al. (p. 1543) analyze what it takes to be an obligate biotroph in barley powdery mildew, and Baxter et al. (p. 1549) ask a similar question for a natural pathogen of Arabidopsis. Schirawski et al. (p. 1546) compared genomes of maize pathogens to identify virulence determinants. Better knowledge of what in a genome makes a pathogen efficient and deadly is likely to be useful for improving agricultural crop management and breeding. A group of papers analyzes pathogen genomes to find the roots of virulence, opportunism, and life-style determinants. Powdery mildews are phytopathogens whose growth and reproduction are entirely dependent on living plant cells. The molecular basis of this life-style, obligate biotrophy, remains unknown. We present the genome analysis of barley powdery mildew, Blumeria graminis f.sp. hordei (Blumeria), as well as a comparison with the analysis of two powdery mildews pathogenic on dicotyledonous plants. These genomes display massive retrotransposon proliferation, genome-size expansion, and gene losses. The missing genes encode enzymes of primary and secondary metabolism, carbohydrate-active enzymes, and transporters, probably reflecting their redundancy in an exclusively biotrophic life-style. Among the 248 candidate effectors of pathogenesis identified in the Blumeria genome, very few (less than 10) define a core set conserved in all three mildews, suggesting that most effectors represent species-specific adaptations.

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.

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 AVRa10 and AVRk1 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, AVRa10 and AVRk1 encode proteins that lack secretion signal peptides and enhance infection success on susceptible host plant cells. AVRa10 and AVRk1 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.

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.

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.

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.

Of genes and genomes, needles and haystacks: Blumeria graminis and functionality

SUMMARY Here, we consider the barley powdery mildew fungus, Blumeria graminis (DC Speer) f.sp. hordei (Marchal), and review recent research which has added to our understanding of the biology and molecular biology which underpins the asexual life cycle of this potentially devastating pathogen. We focus on the early stages of the host-pathogen interaction and report current understanding in the areas of leaf perception, fungal signal transduction and host-imposed oxidative stress management. Through this, it is becoming increasingly clear how closely and subtly both sides of the relationship are regulated. Collectively, however, this review highlights the high degree of complexity in working with an obligate parasite. Our experiences suggest that we would make more efficient progress towards understanding the basis of susceptibility and resistance to this true obligate biotroph if its genome sequence was available.

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.

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.

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.