Fungal Molecular Plant-Microbe Interactions

Mutational Analysis of AREA, a Transcriptional Activator Mediating Nitrogen Metabolite Repression in Aspergillus nidulans and a Member of the “Streetwise” GATA Family of Transcription Factors

Richard A. Wilson et al. — Fri, 25 May 2012 12:26:08 PDT

Details a mutational analysis of AREA, a transcriptional activator mediating nitrogen metabolite repression in Aspergillus nidulans and a member of the “streetwise” GATA family of transcription factors.

Cultivar-Dependent Expression of a Maize Lipoxygenase Responsive to Seed Infesting Fungi

Richard A. Wilson et al. — Fri, 25 May 2012 12:14:07 PDT

Maize kernels are highly susceptible to Aspergillus spp. infection and aflatoxin (AF) contamination. Fatty acid signaling molecules appear to mediate the plant–fungal interaction by affecting the growth, development, and AF production of the fungus. In particular, fatty acid derivatives of the plant lipoxygenase (LOX) pathway are implicated in the Aspergillus spp.-seed interaction. The 9(S)-hydroperoxide derivative of linoleic acid promotes transcription of AF genes, whereas the 13(S)-hydroperoxide derivative decreases AF gene expression and production; both are sporulation factors. Our goal was to identify LOX genes responsive to Aspergillus spp. colonization and determine their specificities, 9(S)- or 13(S)-. Screening maize LOX expressed sequence tags (ESTs) identified one clone, cssap 92, which is highly expressed in Aspergillus spp.-infected seed susceptible to AF contamination and repressed in lines with resistance to AF contamination. The accumulation of cssap 92 transcript was similar during Fusarium spp. infection. The cDNA clone has 94% identity to the previously described L2 LOX gene from maize. Product-specificity analysis of the CSSAP 92 protein shows that it preferentially adds oxygen to carbon 9 of linoleic acid. Because 9(S)-hydroperoxy linoleic acid has been implicated as an aflatoxin-signaling molecule, it is possible that cssap 92 could be used as a biomarker that is indicative of AF resistance in maize lines.

Relationship between Secondary Metabolism and Fungal Development

Ana M. Calvo et al. — Fri, 25 May 2012 12:06:17 PDT

Details findings related to the relationship between secondary metabolism and fungal development.

Two Δ9-Stearic Acid Desaturases Are Required for Aspergillus nidulans Growth and Development

Richard A. Wilson et al. — Fri, 25 May 2012 12:00:06 PDT

Unsaturated fatty acids are important constituents of all cell membranes and are required for normal growth. In the filamentous fungus Aspergillus nidulans, unsaturated fatty acids and their derivatives also influence asexual (conidial) and sexual (ascospore) sporulation processes. To investigate the relationship between fatty acid metabolism and fungal development, we disrupted the A. nidulans sdeA and sdeB genes, both encoding Δ9-stearic acid desaturases responsible for the conversion of palmitic acid (16:0) and stearic acid (18:0) to palmitoleic acid (16:1) and oleic acid (18:1). The effects of sdeA deletion on development were profound, such that growth, conidial and ascospore production were all reduced at 22 and 37°C. Total fatty acid content was increased over 3-fold in the ΔsdeA strain, reflected in up-regulation of the expression of the fasA gene encoding the a chain of the fatty acid synthase, compared to wild type. Stearic acid accumulated approximately 3-fold compared to wild type in the ΔsdeA strain, while unsaturated fatty acid production was decreased. In contrast, disruption of sdeB reduced fungal growth and conidiation at 22°C, but did not affect these processes at 37°C compared to wild type. Interestingly, ascospore production was increased at 37°C for ΔsdeB compared to wild type. Total fatty acid content was not increased in this strain, although stearic acid accumulated 2-fold compared to wild type, and unsaturated fatty acid production was decreased. Combining the ΔsdeA and ΔsdeB alleles created a synthetic lethal strain requiring the addition of oleic acid to the medium for a modicum of growth. Taken together, our results suggest a role for sdeA in growth and development at all temperatures, while sdeB is involved in growth and development at lower temperatures.

Characterization of the Aspergillus parasiticus Δ12-Desaturase Gene: A Role for Lipid Metabolism in the Aspergillus-Seed Interaction

Richard A. Wilson et al. — Fri, 25 May 2012 11:48:36 PDT

In the mycotoxigenic oilseed pathogens Aspergillus flavus and Aspergillus parasiticus and the model filamentous fungus Aspergillus nidulans, unsaturated fatty acids and their derivatives act as important developmental signals that affect asexual conidiospore, sexual ascospore and/or sclerotial development. To dissect the relationship between lipid metabolism and fungal development, an A. parasiticus Δ¹²-desaturase mutant that was unable to convert oleic acid to linoleic acid and was thus impaired in polyunsaturated fatty acid biosynthesis was generated. The Δ¹²-desaturase mutant demonstrates delayed spore germination, a twofold reduction in growth, a reduced level of conidiation and complete loss of sclerotial development, compared to the wild-type. Host colonization is impaired, as reflected by a decrease in conidial production on live peanut and corn seed by the mutant compared to the wild-type. Similarly, the previously isolated A. nidulans Δ¹²-desaturase mutant has reduced colonization capabilities compared to the wild-type. Therefore, desaturation mutants display a key requisite that affords a genetic solution to oilseed crop contamination by mycotoxigenic Aspergillus species: a reduction in the production of conidia, the infectious particle of the pathogenic aspergilli.

An NADPH-Dependent Genetic Switch Regulates Plant Infection by the Rice Blast Fungus

Richard A. Wilson et al. — Fri, 25 May 2012 11:36:16 PDT

To cause rice blast disease, the fungus Magnaporthe oryzae breaches the tough outer cuticle of the rice leaf by using specialized infection structures called appressoria. These cells allow the fungus to invade the host plant and proliferate rapidly within leaf tissue. Here, we show that a unique NADPH-dependent genetic switch regulates plant infection in response to the changing nutritional and redox conditions encountered by the pathogen. The biosynthetic enzyme trehalose-6-phosphate synthase (Tps1) integrates control of glucose-6-phosphate metabolism and nitrogen source utilization by regulating the oxidative pentose phosphate pathway, the generation of NADPH, and the activity of nitrate reductase. We report that Tps1 directly binds to NADPH and, thereby, regulates a set of related transcriptional corepressors, comprising three proteins, Nmr1, Nmr2, and Nmr3, which can each bind NADP. Targeted deletion of any of the Nmr-encoding genes partially suppresses the nonpathogenic phenotype of a Δtps1 mutant. Tps1-dependentNmr corepressors control the expression of a set of virulence-associated genes that are derepressed during appressorium-mediated plant infection. When considered together, these results suggest that initiation of rice blast disease by M. oryzae requires a regulatory mechanism involving an NADPH sensor protein, Tps1, a set of NADP-dependent transcriptional corepressors, and the nonconsuming interconversion ofNADPHandNADPacting as signal transducer.

Fungal Virulence and Development Is Regulated by Alternative Pre-mRNA 3′End Processing in Magnaporthe oryzae

Marina Franceschetti et al. — Fri, 25 May 2012 11:27:21 PDT

RNA-binding proteins play a central role in post-transcriptional mechanisms that control gene expression. Identification of novel RNA-binding proteins in fungi is essential to unravel post-transcriptional networks and cellular processes that confer identity to the fungal kingdom. Here, we carried out the functional characterisation of the filamentous fungus-specific RNAbinding protein RBP35 required for full virulence and development in the rice blast fungus. RBP35 contains an N-terminal RNA recognition motif (RRM) and six Arg-Gly-Gly tripeptide repeats. Immunoblots identified two RBP35 protein isoforms that show a steady-state nuclear localisation and bind RNA in vitro. RBP35 coimmunoprecipitates in vivo with Cleavage Factor I (CFI) 25 kDa, a highly conserved protein involved in polyA site recognition and cleavage of pre-mRNAs. Several targets of RBP35 have been identified using transcriptomics including 14-3-3 pre-mRNA, an important integrator of environmental signals. In Magnaporthe oryzae, RBP35 is not essential for viability but regulates the length of 3'UTRs of transcripts with developmental and virulence-associated functions. The Δrbp35 mutant is affected in the TOR (target of rapamycin) signaling pathway showing significant changes in nitrogen metabolism and protein secretion. The lack of clear RBP35 orthologues in yeast, plants and animals indicates that RBP35 is a novel auxiliary protein of the polyadenylation machinery of filamentous fungi. Our data demonstrate that RBP35 is the fungal equivalent of metazoan CFI 68 kDa and suggest the existence of 3'end processing mechanisms exclusive to the fungal kingdom.

Under Pressure: Investigating the Biology of Plant Infection by Magnaporthe oryza

Nicholas J. Talbot et al. — Fri, 25 May 2012 10:59:41 PDT

The filamentous fungus Magnaporthe oryzae causes rice blast, the most serious disease of cultivated rice. Cellular differentiation of M. oryzae forms an infection structure called the appressorium, which generates enormous cellular turgor that is sufficient to rupture the plant cuticle. Here, we show how functional genomics approaches are providing new insight into the genetic control of plant infection by M. oryzae. We also look ahead to the key questions that need to be addressed to provide a better understanding of the molecular processes that lead to plant disease and the prospects for sustainable control of rice blast.

The Sugar Sensor, Trehalose-6-Phosphate Synthase (Tps1), Regulates Primary and Secondary Metabolism during Infection by the Rice Blast Fungus: Will Magnaporthe oryzae’s “Sweet Tooth” become Its “Achilles’ Heel”?

Jessie Fernandez et al. — Fri, 25 May 2012 10:54:30 PDT

Rice blast disease is considered one of the most serious diseases of cultivated rice and is mediated by the causal agent, Magnaporthe oryzae. During infection, dome-shaped fungal cells, called appressoria, form on the surface of the leaf and generate turgor through the accumulation of glycerol. This enormous pressure is directed down onto a thin penetration hypha emerging from the base of the cell, forcing it through the surface of the rice leaf and allowing fungal colonization of the plant interior. The non-reducing disaccharide, trehalose, is present in conidia of M. oryzae and is mobilized during appressorium formation. The first step in trehalose biosynthesis involves trehalose-6-phosphate synthase (Tps1), and deletion of the TPS1 gene in M. oryzae abolishes its ability to cause disease. This loss of pathogenicity was thought to be due to the role trehalose might play in turgor generation in the appressorium, or from the loss of the trehalose intermediate, trehalose-6-phosphate, a known signalling molecule in other organisms. However, subsequent analysis determined that, in M. oryzae, it is the Tps1 protein itself that is a central regulator of plant infection. Here, we discuss how the role of trehalose metabolism in M. oryzae development was determined to differ from other eukaryotes and show how, independent of its biosynthetic role, Tps1 functions as a sugar sensor to integrate carbon and nitrogen metabolism and regulate a subset of primary and secondary metabolic pathways, such as the oxidative pentose phosphate pathway and pigment formation, respectively, during plant colonization. This is a critical role that allows the fungus to adapt to the nutritional and redox conditions encountered in the plant cell and establish disease.

Fungal Physiology: A Future Perspective

Richard A. Wilson et al. — Fri, 25 May 2012 10:51:06 PDT

The study of fungal physiology is set to change dramatically in the next few years as highly scalable technologies are deployed allowing accurate measurement and identification of metabolites, proteins and transcripts within cells. The advent of next-generation DNA-sequencing technologies will also provide genome sequence information from large numbers of industrially relevant and pathogenic fungal species, and allow comparative genome analysis between strains and populations of fungi. When coupled with advances in gene functional analysis, protein-protein interaction studies, live cell imaging and mathematical modelling, this promises a step-change in our understanding of how fungal cells operate as integrated dynamic living systems

Oxygenase Coordination Is Required for Morphological Transition and the Host-Fungus Interaction of Aspergillus flavus

Sigal Horowitz Brown et al. — Fri, 25 May 2012 10:48:27 PDT

Oxylipins, a class of oxygenase-derived unsaturated fatty acids, are important signal molecules in many biological systems. Recent characterization of an Aspergillus flavus lipoxygenase gene, lox, revealed its importance in maintaining a density-dependent morphology switch from sclerotia to conidia as population density increased. Here, we present evidence for the involvement of four more oxylipingenerating dioxygenases (PpoA, PpoB, PpoC, and PpoD) in A. flavus density-dependent phenomena and the effects of loss of these genes on aflatoxin production and seed colonization. Although several single mutants showed alterations in the sclerotia-to-conidia switch, the major effect was observed in a strain downregulated for all five oxygenases (invert repeat transgene [IRT] strain IRT4 = ppoA, ppoB, ppoC, ppoD, and lox). In strain IRT4, sclerotia production was increased up to 500-fold whereas conidiation was decreased down to 100-fold and the strain was unable to switch into conidial production. Aflatoxin (AF) production for all mutant strains and the wild type was greatest at low population densities and absent in high populations except for strain IRT4, which consistently produced high levels of the mycotoxin. Growth on host seed by both IRT4 and IRT2 (downregulated in ppoA, ppoB, and ppoD) was marked by decreased conidial but increased AF production. We propose that A. flavus oxygenases and the oxylipins they produce act in a highly interdependent network with some redundancy of biological function. These studies provide substantial evidence for oxylipin-based mechanisms in governing fungus-seed interactions and in regulating a coordinated quorum-sensing mechanism in A. flavus.

Fundamental Contribution of β-Oxidation to Polyketide Mycotoxin Production In Planta

Lori A. Maggio-Hall et al. — Fri, 25 May 2012 10:42:06 PDT

Seed contamination with polyketide mycotoxins, including aflatoxin (AF) and sterigmatocystin (ST) produced by Aspergillus spp., is an agricultural, economic, and medical issue worldwide. Acetyl-CoA, the fundamental building block of all known fungal polyketides, is generated by a large number of biochemical pathways, including β-oxidation of fatty acids and glycolysis of sugars. We present several lines of evidence to support a major role for seed fatty acids in formation of AF and ST in A. flavus, A. parasiticus, and A. nidulans. Aspergillus strains exhibiting canonical signs of oleic acid–induced peroxisome proliferation, including increased catalase activity, β-oxidation gene expression, and peroxisomal clustering, also exhibited a marked increase in toxin gene expression and biosynthesis. Furthermore, microscopic observations showed that the ST and AF precursor norsolorinic acid accumulated in peroxisomes of all three Aspergilli. While a peroxisomal β-oxidation mutation eliminated oleic acid–induced increases in ST in A. nidulans, a mitochondrial β-oxidation mutation played a larger role in eliminating ST formation on oatmeal medium and on live corn kernels, implicating a fundamental role for both peroxisomal and mitochondrial β-oxidation in toxin production.

Deletion of the Δ12-Oleic Acid Desaturase Gene of a Nonaflatoxigenic Aspergillus parasiticus Field Isolate Affects Conidiation and Sclerotial Development

Perng Kuang Chang et al. — Fri, 25 May 2012 10:34:48 PDT

Aims: To investigate how linoleic acid affects conidial production and sclerotial development in a strictly mitotic Aspergillus parasiticus field isolate as related to improving biocompetitivity of atoxigenic Aspergillus species.

Methods and Results: We disrupted A. parasiticus Δ12-oleic acid desaturase gene (odeA) responsible for the conversion of oleic acid to linoleic acid. We examined conidiation and sclerotial development of SRRC 2043 and three isogenic mutant strains deleted for the odeA gene (DodeA), either with or without supplementing linoleic acid, on one complex potato dextrose agar (PDA) medium and on two defined media: nitrate-containing Czapek agar (CZ) and Cove’s ammonium medium (CVN). The ΔodeA mutants produced less conidia than the parental strain on all media. Linoleic acid supplementation (as sodium linoleate at 0.3 and 1.2 mg ml^-1) restored the ΔodeA conidial production comparable to or exceeding the unsupplemented parental level, and the effect was medium dependent, with the highest increase on CVN and the least on PDA. SRRC 2043 and the DodeA mutants were unable to produce sclerotia on CVN. On unsupplemented PDA and CZ, ΔodeA sclerotial mass was comparable to that of SRRC 2043, but sclerotial number increased significantly to two- to threefold. Supplementing linoleic acid to media, in general, tended to decrease wild type and ΔodeA sclerotial mass and sclerotial number.

Conclusions: Linoleic acid stimulates conidial production but has an inhibitory effect on sclerotial development. The relationship between the two processes in A. parasiticus is complex and affected by multiple factors, such as fatty acid composition and nitrogen source.

Significance and Impact of Study: Conditions that promote sclerotial development differ from those required to promote maximum conidial production. Manipulation of content and availability of linoleic acid at different fungal growth phases might optimize conidial and sclerotial production hence increasing the efficacy of biocompetitive Aspergillus species.

Principles of Carbon Catabolite Repression in the Rice Blast Fungus: Tps1, Nmr1-3, and a MATE–Family Pump Regulate Glucose Metabolism during Infection

Jessie Fernandez et al. — Fri, 25 May 2012 10:06:04 PDT

Understanding the genetic pathways that regulate how pathogenic fungi respond to their environment is paramount to developing effective mitigation strategies against disease. Carbon catabolite repression (CCR) is a global regulatory mechanism found in a wide range of microbial organisms that ensures the preferential utilization of glucose over less favourable carbon sources, but little is known about the components of CCR in filamentous fungi. Here we report three new mediators of CCR in the devastating rice blast fungus Magnaporthe oryzae: the sugar sensor Tps1, the Nmr1-3 inhibitor proteins, and the multidrug and toxin extrusion (MATE)-family pump, Mdt1. Using simple plate tests coupled with transcriptional analysis, we show that Tps1, in response to glucose-6-phosphate sensing, triggers CCR via the inactivation of Nmr1-3. In addition, by dissecting the CCR pathway using Agrobacterium tumefaciens-mediated mutagenesis, we also show that Mdt1 is an additional and previously unknown regulator of glucose metabolism. Mdt1 regulates glucose assimilation downstream of Tps1 and is necessary for nutrient utilization, sporulation, and pathogenicity. This is the first functional characterization of a MATE-family protein in filamentous fungi and the first description of a MATE protein in genetic regulation or plant pathogenicity. Perturbing CCR in Δtps1 and MDT1 disruption strains thus results in physiological defects that impact pathogenesis, possibly through the early expression of cell wall-degrading enzymes. Taken together, the importance of discovering three new regulators of carbon metabolism lies in understanding how M. oryzae and other pathogenic fungi respond to nutrient availability and control development during infection.