Inferring Roles in Defense from Metabolic Allocation of Rice Diterpenoids.

Among their responses to microbial infection, plants deploy an arsenal of natural antibiotic products. Historically these have been identified on the basis of their antibiotic activity in vitro, which leaves open the question of their relevance to defense in planta. The vast majority of such natural products from the important crop plant rice (Oryza sativa) are diterpenoids whose biosynthesis proceeds via either ent- or syn-copalyl diphosphate (CPP) intermediates, which were isolated on the basis of their antibiotic activity against the fungal blast pathogen Magnaporthe oryzae However, rice plants in which the gene for the syn-CPP synthase Os-CPS4 is knocked out do not exhibit increased susceptibility to M. oryzae Here, we show that knocking out or knocking down Os-CPS4 actually decreases susceptibility to the bacterial leaf blight pathogen Xanthomonas oryzae By contrast, genetic manipulation of the gene for the ent-CPP synthase Os-CPS2 alters susceptibility to both M. oryzae and X. oryzae Despite the secretion of diterpenoids dependent on Os-CPS2 or Os-CPS4 from roots, neither knockout exhibited significant changes in the composition of their rhizosphere bacterial communities. Nevertheless, rice plants allocate substantial metabolic resources toward syn- as well as ent-CPP derived diterpenoids upon infection/induction. Further investigation revealed that Os-CPS4 plays a role in fungal non-host disease resistance. Thus, examination of metabolic allocation provides important clues into physiological function.

Tpc1 is an important Zn(II)2Cys6 transcriptional regulator required for polarized growth and virulence in the rice blast fungus.

The establishment of polarity is a critical process in pathogenic fungi, mediating infection-related morphogenesis and host tissue invasion. Here, we report the identification of TPC1 (Transcription factor for Polarity Control 1), which regulates invasive polarized growth in the rice blast fungus Magnaporthe oryzae. TPC1 encodes a putative transcription factor of the fungal Zn(II)2Cys6 family, exclusive to filamentous fungi. Tpc1-deficient mutants show severe defects in conidiogenesis, infection-associated autophagy, glycogen and lipid metabolism, and plant tissue colonisation. By tracking actin-binding proteins, septin-5 and autophagosome components, we show that Tpc1 regulates cytoskeletal dynamics and infection-associated autophagy during appressorium-mediated plant penetration. We found that Tpc1 interacts with Mst12 and modulates its DNA-binding activity, while Tpc1 nuclear localisation also depends on the MAP kinase Pmk1, consistent with the involvement of Tpc1 in this signalling pathway, which is critical for appressorium development. Importantly, Tpc1 directly regulates NOXD expression, the p22phox subunit of the fungal NADPH oxidase complex via an interaction with Mst12. Tpc1 therefore controls spatial and temporal regulation of cortical F-actin through regulation of the NADPH oxidase complex during appressorium re-polarisation. Consequently, Tpc1 is a core developmental regulator in filamentous fungi, linking the regulated synthesis of reactive oxygen species and the Pmk1 pathway, with polarity control during host invasion.

Virulence- and signaling-associated genes display a preference for long 3'UTRs during rice infection and metabolic stress in the rice blast fungus.

Generation of mRNA isoforms by alternative polyadenylation (APA) and their involvement in regulation of fungal cellular processes, including virulence, remains elusive. Here, we investigated genome-wide polyadenylation site (PAS) selection in the rice blast fungus to understand how APA regulates pathogenicity. More than half of Magnaporthe oryzae transcripts undergo APA and show novel motifs in their PAS region. Transcripts with shorter 3'UTRs are more stable and abundant in polysomal fractions, suggesting they are being translated more efficiently. Importantly, rice colonization increases the use of distal PASs of pathogenicity genes, especially those participating in signalling pathways like 14-3-3B, whose long 3'UTR is required for infection. Cleavage factor I (CFI) Rbp35 regulates expression and distal PAS selection of virulence and signalling-associated genes, tRNAs and transposable elements, pointing its potential to drive genomic rearrangements and pathogen evolution. We propose a noncanonical PAS selection mechanism for Rbp35 that recognizes UGUAH, unlike humans, without CFI25. Our results showed that APA controls turnover and translation of transcripts involved in fungal growth and environmental adaptation. Furthermore, these data provide useful information for enhancing genome annotations and for cross-species comparisons of PASs and PAS usage within the fungal kingdom and the tree of life.

Molecular characterization of a novel ssRNA ourmia-like virus from the rice blast fungus Magnaporthe oryzae.

In this study we characterize a novel positive and single stranded RNA (ssRNA) mycovirus isolated from the rice field isolate of Magnaporthe oryzae Guy11. The ssRNA contains a single open reading frame (ORF) of 2,373 nucleotides in length and encodes an RNA-dependent RNA polymerase (RdRp) closely related to ourmiaviruses (plant viruses) and ourmia-like mycoviruses. Accordingly, we name this virus Magnaporthe oryzae ourmia-like virus 1 (MOLV1). Although phylogenetic analysis suggests that MOLV1 is closely related to ourmia and ourmia-like viruses, it has some features never reported before within the Ourmiavirus genus. 3' RLM-RACE (RNA ligase-mediated rapid amplification of cDNA ends) and extension poly(A) tests (ePAT) suggest that the MOLV1 genome contains a poly(A) tail whereas the three cytosine and the three guanine residues present in 5' and 3' untranslated regions (UTRs) of ourmia viruses are not observed in the MOLV1 sequence. The discovery of this novel viral genome supports the hypothesis that plant pathogenic fungi may have acquired this type of viruses from their host plants.

Light-dependent gene activation in Aspergillus nidulans is strictly dependent on phytochrome and involves the interplay of phytochrome and white collar-regulated histone H3 acetylation.

The ability for light sensing is found from bacteria to humans but relies only on a small number of evolutionarily conserved photoreceptors. A large number of fungi react to light, mostly to blue light. Aspergillus nidulans also responds to red light using a phytochrome light sensor, FphA, for the control of hundreds of light-regulated genes. Here, we show that photoinduction of one light-induced gene, ccgA, occurs mainly through red light. Induction strictly depends on phytochrome and its histidine-kinase activity. Full light activation also depends on the Velvet protein, VeA. This putative transcription factor binds to the ccgA promoter in an fphA-dependent manner but independent of light. In addition, the blue light receptor LreA binds to the ccgA promoter in the dark but is released after blue or red light illumination and together with FphA modulates gene expression through histone H3 modification. LreA interacts with the acetyltransferase GcnE and with the histone deacetylase HdaA. ccgA induction is correlated to an increase of the acetylation level of lysine 9 in histone H3. Our results suggest regulation of red light-induced genes at the transcriptional level involving transcription factor(s) and epigenetic control through modulation of the acetylation level of histone H3.

Fungi, hidden in soil or up in the air: light makes a difference.

Light is one of the most important environmental factors for orientation of almost all organisms on Earth. Whereas light sensing is of crucial importance in plants to optimize light-dependent energy conservation, in nonphotosynthetic organisms, the synchronization of biological clocks to the length of a day is an important function. Filamentous fungi may use the light signal as an indicator for the exposure of hyphae to air and adapt their physiology to this situation or induce morphogenetic pathways. Although a yes/no decision appears to be sufficient for the light-sensing function in fungi, most species apply a number of different, wavelength-specific receptors. The core of all receptor types is a chromophore, a low-molecular-weight organic molecule, such as flavin, retinal, or linear tetrapyrrols for blue-, green-, or red-light sensing, respectively. Whereas the blue-light response in fungi is one of the best-studied light responses, all other light-sensing mechanisms are less well studied or largely unknown. The discovery of phytochrome in bacteria and fungi in recent years not only advanced the scientific field significantly, but also had great impact on our view of the evolution of phytochrome-like photoreceptors.

Transcriptional changes in the transition from vegetative cells to asexual development in the model fungus Aspergillus nidulans.

Morphogenesis encompasses programmed changes in gene expression that lead to the development of specialized cell types. In the model fungus Aspergillus nidulans, asexual development involves the formation of characteristic cell types, collectively known as the conidiophore. With the aim of determining the transcriptional changes that occur upon induction of asexual development, we have applied massive mRNA sequencing to compare the expression pattern of 19-h-old submerged vegetative cells (hyphae) with that of similar hyphae after exposure to the air for 5 h. We found that the expression of 2,222 (20.3%) of the predicted 10,943 A. nidulans transcripts was significantly modified after air exposure, 2,035 being downregulated and 187 upregulated. The activation during this transition of genes that belong specifically to the asexual developmental pathway was confirmed. Another remarkable quantitative change occurred in the expression of genes involved in carbon or nitrogen primary metabolism. Genes participating in polar growth or sexual development were transcriptionally repressed, as were those belonging to the HogA/SakA stress response mitogen-activated protein (MAP) kinase pathway. We also identified significant expression changes in several genes purportedly involved in redox balance, transmembrane transport, secondary metabolite production, or transcriptional regulation, mainly binuclear-zinc cluster transcription factors. Genes coding for these four activities were usually grouped in metabolic clusters, which may bring regulatory implications for the induction of asexual development. These results provide a blueprint for further stage-specific gene expression studies during conidiophore development.

Determination of the Mycovirome of a Necrotrophic Fungus.

Next-generation sequencing (NGS) of total RNA has allowed the detection of novel viruses infecting different hosts, such as fungi, increasing our knowledge on virus horizontal transfer events among different hosts, virus diversity, and virus evolution. Here, we describe the detailed protocols for the isolation of the plant pathogenic fungus Botrytis cinerea, from grapevine plants showing symptoms of the mold gray disease, the culture and maintenance of the isolated B. cinerea strains, the extraction of total RNA from B. cinerea strains for NGS, the bioinformatics pipeline designed and followed to detect mycoviruses in the sequenced samples, and the validation of the in silico detected mycoviruses by different approaches.

Construction of a Mycoviral Infectious Clone for Reverse Genetics in Botrytis cinerea.

The most important advances in our understanding of the viral life cycle, such as genome replication, packaging, transmission, and host interactions, have been made via the development of viral infectious full-length clones. Here, we describe the detailed protocols for the construction of an infectious clone derived from Botrytis virus F (BVF), a mycoflexivirus infecting the plant pathogenic fungus Botrytis cinerea, the determination of the complete sequence of the cloned mycovirus, the preparation of fungal protoplasts, and the transfection of protoplasts using transcripts derived from the BVF infectious clone.

Phycomyces MADB interacts with MADA to form the primary photoreceptor complex for fungal phototropism.

The fungus Phycomyces blakesleeanus reacts to environmental signals, including light, gravity, touch, and the presence of nearby objects, by changing the speed and direction of growth of its fruiting body (sporangiophore). Phototropism, growth toward light, shares many features in fungi and plants but the molecular mechanisms remain to be fully elucidated. Phycomyces mutants with altered phototropism were isolated approximately 40 years ago and found to have mutations in the mad genes. All of the responses to light in Phycomyces require the products of the madA and madB genes. We showed that madA encodes a protein similar to the Neurospora blue-light photoreceptor, zinc-finger protein WC-1. We show here that madB encodes a protein similar to the Neurospora zinc-finger protein WC-2. MADA and MADB interact to form a complex in yeast 2-hybrid assays and when coexpressed in E. coli, providing evidence that phototropism and other responses to light are mediated by a photoresponsive transcription factor complex. The Phycomyces genome contains 3 genes similar to wc-1, and 4 genes similar to wc-2, many of which are regulated by light in a madA or madB dependent manner. We did not detect any interactions between additional WC proteins in yeast 2-hybrid assays, which suggest that MADA and MADB form the major photoreceptor complex in Phycomyces. However, the presence of multiple wc genes in Phycomyces may enable perception across a broad range of light intensities, and may provide specialized photoreceptors for distinct photoresponses.

Genomic analysis of the basal lineage fungus Rhizopus oryzae reveals a whole-genome duplication.

Rhizopus oryzae is the primary cause of mucormycosis, an emerging, life-threatening infection characterized by rapid angioinvasive growth with an overall mortality rate that exceeds 50%. As a representative of the paraphyletic basal group of the fungal kingdom called "zygomycetes," R. oryzae is also used as a model to study fungal evolution. Here we report the genome sequence of R. oryzae strain 99-880, isolated from a fatal case of mucormycosis. The highly repetitive 45.3 Mb genome assembly contains abundant transposable elements (TEs), comprising approximately 20% of the genome. We predicted 13,895 protein-coding genes not overlapping TEs, many of which are paralogous gene pairs. The order and genomic arrangement of the duplicated gene pairs and their common phylogenetic origin provide evidence for an ancestral whole-genome duplication (WGD) event. The WGD resulted in the duplication of nearly all subunits of the protein complexes associated with respiratory electron transport chains, the V-ATPase, and the ubiquitin-proteasome systems. The WGD, together with recent gene duplications, resulted in the expansion of multiple gene families related to cell growth and signal transduction, as well as secreted aspartic protease and subtilase protein families, which are known fungal virulence factors. The duplication of the ergosterol biosynthetic pathway, especially the major azole target, lanosterol 14alpha-demethylase (ERG11), could contribute to the variable responses of R. oryzae to different azole drugs, including voriconazole and posaconazole. Expanded families of cell-wall synthesis enzymes, essential for fungal cell integrity but absent in mammalian hosts, reveal potential targets for novel and R. oryzae-specific diagnostic and therapeutic treatments.

Construction and Characterization of a Botrytis Virus F Infectious Clone.

Botrytis virus F (BVF) is a positive-sense, single-stranded RNA (+ssRNA) virus within the Gammaflexiviridae family of the plant-pathogenic fungus Botrytis cinerea. In this study, the complete sequence of a BVF strain isolated from B. cinerea collected from grapevine fields in Spain was analyzed. This virus, in this work BVF-V448, has a genome of 6827 nt in length, excluding the poly(A) tail, with two open reading frames encoding an RNA dependent RNA polymerase (RdRP) and a coat protein (CP). The 5'- and 3'-terminal regions of the genome were determined by rapid amplification of cDNA ends (RACE). Furthermore, a yet undetected subgenomic RNA species in BVF-V448 was identified, indicating that the CP is expressed via 3' coterminal subgenomic RNAs (sgRNAs). We also report the successful construction of the first BVF full-length cDNA clone and synthesized in vitro RNA transcripts using the T7 polymerase, which could efficiently transfect two different strains of B. cinerea, B05.10 and Pi258.9. The levels of growth in culture and virulence on plants of BVF-V448 transfected strains were comparable to BVF-free strains. The infectious clones generated in this work provide a useful tool for the future development of an efficient BVF foreign gene expression vector and a virus-induced gene silencing (VIGS) vector as a biological agent for the control of B. cinerea.

Novel Mycoviruses Discovered in the Mycovirome of a Necrotrophic Fungus.

Botrytis cinerea is one of the most important plant-pathogenic fungus. Products based on microorganisms can be used in biocontrol strategies alternative to chemical control, and mycoviruses have been explored as putative biological agents in such approaches. Here, we have explored the mycovirome of B. cinerea isolates from grapevine of Italy and Spain to increase the knowledge about mycoviral diversity and evolution, and to search for new widely distributed mycoviruses that could be active ingredients in biological products to control this hazardous fungus. A total of 248 B. cinerea field isolates were used for our metatranscriptomic study. Ninety-two mycoviruses were identified: 62 new mycoviral species constituting putative novel viral genera and families. Of these mycoviruses, 57 had a positive-sense single-stranded RNA (ssRNA) genome, 19 contained a double-stranded RNA (dsRNA) genome, 15 had a negative-sense ssRNA genome, and 1 contained a single-stranded DNA (ssDNA) genome. In general, ssRNA mycoviruses were widely distributed in all sampled regions, the ssDNA mycovirus was more frequently found in Spain, and dsRNA mycoviruses were scattered in some pools of both countries. Some of the identified mycoviruses belong to clades that have never been found associated with Botrytis species: Botrytis-infecting narnaviruses; alpha-like, umbra-like, and tymo-like ssRNA+ mycoviruses; trisegmented ssRNA- mycovirus; bisegmented and tetrasegmented dsRNA mycoviruses; and finally, an ssDNA mycovirus. Among the results obtained in this massive mycovirus screening, the discovery of novel bisegmented viruses, phylogenetically related to narnaviruses, is remarkable.IMPORTANCE The results obtained here have expanded our knowledge of mycoviral diversity, horizontal transfers, and putative cross-kingdom events. To date, this study presents the most extensive and wide diversity collection of mycoviruses infecting the necrotrophic fungus B. cinerea The collection included all types of mycoviruses, with dsRNA, ssRNA+, ssRNA-, and ssDNA genomes, most of which were discovered here, and some of which were previously reported as infecting B. cinerea or other plant-pathogenic fungi. Some of these mycoviruses are reported for the first time here associated with B. cinerea, as a trisegmented ssRNA- mycovirus and as an ssDNA mycovirus, but even more remarkablly, we also describe here four novel bisegmented viruses (binarnaviruses) not previously described in nature. The present findings significantly contribute to general knowledge in virology and more particularly in the field of mycovirology.

Spotlight on Aspergillus nidulans photosensory systems.

Aspergilli are ubiquitous soil-borne fungi growing within or on the surface of numerous organic substrates. Growth within a substrate or growth on the surface correlates to different growth conditions for the hyphae due to significant changes in oxygen or reactive oxygen species levels and variations in humidity or temperature. The production of air-borne spores is supported by the substrate-air interphase and also requires a sensing system to adapt appropriately. Here we focus on light as important parameter for the mycelium to discriminate between different habitats. The fungal 'eye' includes several light sensors which react to a broad plethora of wavelengths. Aspergillus nidulans light receptors comprise a phytochrome for red-light sensing, white collar-like blue-light signaling proteins, a putative green-light sensing opsin and a cryptochrome/photolyase as distinct sensory systems. Red- and blue-light receptors are assembled into a light-sensing protein complex. Light receptors transmit their signal to a number of other regulatory proteins including a bridging protein, VeA, as part of a trimeric complex. VeA plays a central role in the balance of asexual and sexual development and in the coordination of morphogenesis and secondary metabolism.

Genome-wide polyadenylation site mapping datasets in the rice blast fungus Magnaporthe oryzae.

Polyadenylation plays an important role in gene regulation, thus affecting a wide variety of biological processes. In the rice blast fungus Magnaporthe oryzae the cleavage factor I protein Rpb35 is required for pre-mRNA polyadenylation and fungal virulence. Here we present the bioinformatic approach and output data related to a global survey of polyadenylation site usage in M. oryzae wild-type and Δrbp35 strains under a variety of nutrient conditions, some of which simulate the conditions experienced by the fungus during part of its infection cycle.

Expansion of Signal Transduction Pathways in Fungi by Extensive Genome Duplication.

Plants and fungi use light and other signals to regulate development, growth, and metabolism. The fruiting bodies of the fungus Phycomyces blakesleeanus are single cells that react to environmental cues, including light, but the mechanisms are largely unknown [1]. The related fungus Mucor circinelloides is an opportunistic human pathogen that changes its mode of growth upon receipt of signals from the environment to facilitate pathogenesis [2]. Understanding how these organisms respond to environmental cues should provide insights into the mechanisms of sensory perception and signal transduction by a single eukaryotic cell, and their role in pathogenesis. We sequenced the genomes of P. blakesleeanus and M. circinelloides and show that they have been shaped by an extensive genome duplication or, most likely, a whole-genome duplication (WGD), which is rarely observed in fungi [3-6]. We show that the genome duplication has expanded gene families, including those involved in signal transduction, and that duplicated genes have specialized, as evidenced by differences in their regulation by light. The transcriptional response to light varies with the developmental stage and is still observed in a photoreceptor mutant of P. blakesleeanus. A phototropic mutant of P. blakesleeanus with a heterozygous mutation in the photoreceptor gene madA demonstrates that photosensor dosage is important for the magnitude of signal transduction. We conclude that the genome duplication provided the means to improve signal transduction for enhanced perception of environmental signals. Our results will help to understand the role of genome dynamics in the evolution of sensory perception in eukaryotes.

A relationship between carotenoid accumulation and the distribution of species of the fungus Neurospora in Spain.

The ascomycete fungus Neurospora is present in many parts of the world, in particular in tropical and subtropical areas, where it is found growing on recently burned vegetation. We have sampled the Neurospora population across Spain. The sampling sites were located in the region of Galicia (northwestern corner of the Iberian peninsula), the province of Cáceres, the city of Seville, and the two major islands of the Canary Islands archipelago (Tenerife and Gran Canaria, west coast of Africa). The sites covered a latitude interval between 27.88° and 42.74°. We have identified wild-type strains of N. discreta, N. tetrasperma, N. crassa, and N. sitophila and the frequency of each species varied from site to site. It has been shown that after exposure to light Neurospora accumulates the orange carotenoid neurosporaxanthin, presumably for protection from UV radiation. We have found that each Neurospora species accumulates a different amount of carotenoids after exposure to light, but these differences did not correlate with the expression of the carotenogenic genes al-1 or al-2. The accumulation of carotenoids in Neurospora shows a correlation with latitude, as Neurospora strains isolated from lower latitudes accumulate more carotenoids than strains isolated from higher latitudes. Since regions of low latitude receive high UV irradiation we propose that the increased carotenoid accumulation may protect Neurospora from high UV exposure. In support of this hypothesis, we have found that N. crassa, the species that accumulates more carotenoids, is more resistant to UV radiation than N. discreta or N. tetrasperma. The photoprotection provided by carotenoids and the capability to accumulate different amounts of carotenoids may be responsible, at least in part, for the distribution of Neurospora species that we have observed across a range of latitudes.

Regulation of conidiation by light in Aspergillus nidulans.

Light regulates several aspects of the biology of many organisms, including the balance between asexual and sexual development in some fungi. To understand how light regulates fungal development at the molecular level we have used Aspergillus nidulans as a model. We have performed a genome-wide expression analysis that has allowed us to identify >400 genes upregulated and >100 genes downregulated by light in developmentally competent mycelium. Among the upregulated genes were genes required for the regulation of asexual development, one of the major biological responses to light in A. nidulans, which is a pathway controlled by the master regulatory gene brlA. The expression of brlA, like conidiation, is induced by light. A detailed analysis of brlA light regulation revealed increased expression after short exposures with a maximum after 60 min of light followed by photoadaptation with longer light exposures. In addition to brlA, genes flbA-C and fluG are also light regulated, and flbA-C are required for the correct light-dependent regulation of the upstream regulator fluG. We have found that light induction of brlA required the photoreceptor complex composed of a phytochrome FphA, and the white-collar homologs LreA and LreB, and the fluffy genes flbA-C. We propose that the activation of regulatory genes by light is the key event in the activation of asexual development by light in A. nidulans.

Regulation by blue light and heat shock of gene transcription in the fungus Phycomyces: proteins required for photoinduction and mechanism for adaptation to light.

The gene hspA for the heat-shock protein HSP100 is induced by blue light and heat shock in the zygomycete fungus Phycomyces blakesleeanus. We have investigated the molecular details of the regulation of hspA gene transcription. We have cloned 1.9 kb of hspA upstream DNA sequence and identified many DNA segments possibly involved in heat-shock and blue-light regulation. We have identified several gene products required for hspA photoactivation and found that they are also required for mycelial photoresponses, a suggestion for a common signal transduction pathway. In addition, we have found that beta-carotene, or a chemical derivative, is required for hspA gene photoactivation. The activation of hspA after blue light-exposure or a heat shock is transient, suggesting the adaptation to the stimulus. The adaptation of hspA photoactivation seems to be the result of a novel mechanism causing a light-dependent loss of gene transcription. We propose that a reduction in the amount of MADA, a putative flavin-binding zinc-finger protein, in light-exposed mycelia may cause a reduced hspA photoactivation, providing a simple explanation for adaptation to light.