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In this study, we analysed a total of 16 libraries from over 150 grapevine leaf and grape samples infected with Plasmopara viticola (downy mildew of grapevine) to characterise the virome associated to this oomycete. Samples were collected in five distinct regions in Italy and in four different regions in Spain, representative of different pedoclimatic conditions and different grapevine cultivars during 2018 growing season. Due to the metagenomics nature of the samples (containing at least both downy mildew hyphae and spores, and grapevine cells residues), we were able to assemble several plant viruses and a few possible novel plant virus genomes with our in silico analysis. We detected several plant virus variants already reported in grapevine, and a putative new ilarvirus previously unreported in grapevine. Furthermore, we characterised three new phenui-like viruses (in the order Bunyavirales), one of which shares some commonalities with plant coguviruses. Finally, we report a new strict association of three viral segments (one flavi-like and two virga-like) that we propose to be a new virus taxon named jivivirus.

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.

Among their responses to microbial infection, plants deploy an arsenal of natural antibiotic products. These historically 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, and 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 knock-out exhibited significant changes in the composition of their rhizosphere bacterial communities. Nevertheless, rice plants allocate substantial metabolic resources towards syn- and 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.

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., Scientific Data, 5, ISSN:2052-4463

Cleavage factor I (CFI) proteins are core components of the polyadenylation machinery that can regulate several steps of mRNA life cycle, including alternative polyadenylation, splicing, export and decay. Here, we describe the regulatory mechanisms that control two fungal CFI protein classes in Magnaporthe oryzae: Rbp35/CfI25 complex and Hrp1. Using mutational, genetic and biochemical studies we demonstrate that cellular concentration of CFI mRNAs is a limited indicator of their protein abundance. Our results suggest that several post-transcriptional mechanisms regulate Rbp35/CfI25 complex and Hrp1 in the rice blast fungus, some of which are also conserved in other ascomycetes. With respect to Rbp35, these include C-terminal processing, RGG-dependent localization and cleavage, C-terminal autoregulatory domain and regulation by an upstream open reading frame of Rbp35-dependent TOR signalling pathway. Our proteomic analyses suggest that Rbp35 regulates the levels of proteins involved in melanin and phenylpropanoids synthesis, among others. The drastic reduction of fungal CFI proteins in carbon-starved cells suggests that the pre-mRNA processing pathway is altered. Our findings uncover broad and multilayer regulatory mechanisms controlling fungal polyadenylation factors, which have profound implications in pre-mRNA maturation. This area of research offers new avenues for fungicide design by targeting fungal-specific proteins that globally affect thousands of mRNAs.

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.

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.

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.

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 ≈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.

Phycomyces blakesleeanus is a filamentous zygomycete fungus that produces striking elongated single cells that extend up to 10 cm into the air, with each such sporangiophore supporting a sphere containing the spores for dispersal. This organism has served as a model for the detection of environmental signals as diverse as light, chemicals, touch, wind, gravity, and adjacent objects. In particular, sporangiophore growth is regulated by light, and it exhibits phototropism by bending toward near-UV and blue wavelengths and away from far-UV wavelengths in a manner that is physiologically similar to plant phototropic responses. The Phycomyces madA mutants were first isolated more than 40 years ago, and they exhibit reduced sensitivity to light. Here, we identify two (duplicated) homologs in the White Collar 1 family of blue-light photoreceptors in Phycomyces . We describe that the madA mutant strains contain point mutations in one of these genes and that these mutations cosegregate with a defect in phototropism after genetic crosses. Thus, the phototropic responses of fungi through madA and plants through phototropin rely on diverse proteins; however, these proteins share a conserved flavin-binding domain for photon detection.

32 p.-6 fig.-2 tab., 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. © 2013, American Society for Microbiology. All Rights Reserved., This work has been supported by the Basque Government through grant (IT393-10) and the Ministerio de Economía y Competitividad (formerly Ministerio de Ciencia e Innovación) through grant (BFU2010-17528) to U.U., grant (BFU2009- 08701) to E.A.E. and grants from the German Science Foundation (DFG Fi 459), the Fonds der Chemischen Industrie, the Baden-Württemberg Stiftung, and the Centre for Functional Nanostructures to R.F. A. G. is now a contract researcher from The University of The Basque Country. J.R. was a postdoctoral fellow of the Ministerio de Ciencia e Innovación. O.E is a contract researcher associated to grant BFU2010- 17528.

The Magnaporthaceae family includes fungal species that cause devastating diseases on cereals and grasses. The causal agent of take-all disease of wheat Gaeumannomyces graminis, the rice blast fungus Magnaporthe oryzae, and Magnaporthe poae which causes the grey leaf spot on turfgrasses, belong to this family. M. poae and G. graminis are considered root pathogens, whereas M. oryzae is found on aerial plant tissues. Remarkably, M. oryzae can also infect roots and distinct mechanisms control its root infection ability compared to leaf colonisation. Since G. graminis and M. poae are genetically intractable, M. oryzae underground infection process can be used to dissect genetic pathways and molecular mechanisms underlying root infection in other members of Magnaporthaceae. Interestingly, M. oryzae root infection process also shares similarities with ancient mycorrhizal associations. Here, we highlight the latest advances on the mechanisms regulating pathogenicity in these economically significant plant pathogens.

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 Ca´ceres, 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 27.88u and 42.74u. 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.

Summary 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.

We cloned and sequenced the Phycomyces hspA gene. The hspA gene product is a 901-amino-acid protein member of the clpB/HSP100 family. HSP100 proteins are ATPases involved in high-temperature tolerance, proteolysis, and protein disaggregation. Phycomyces HSP100 is composed of a domain presumably involved in protein-protein interactions and two ATP-binding domains. The hspA promoter contains three heat-shock elements that are presumably involved in the activation of hspA after heat-shock. In addition, four short sequences are present in the hspA promoter and in the promoter of the photoinducible genes carB and carRA; and these are candidates as binding sites for light-regulated transcription factors. Blue light can increase transcription of the hspA gene 10-fold, with a threshold of 1 J/m2. The threshold for hspA photoactivation is 10(4) times higher than the thresholds for blue-light regulation of sporangiophore development and photocarotenogenesis, which suggests that there are differences in the photosensory systems for gene photoactivation and mycelial photoresponses. A heat-shock of 30 min at 34 degrees C or 42 degrees C increased hspA gene activity 160-fold. The differences in maximum hspA gene transcription by blue light and heatshock suggest the presence of different regulatory mechanisms.

Centro de Biotecnología y Genómica de Plantas (CBGP), 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., J.R.-R. was partially supported by a postdoctoral fellowship from the Severo Ochoa Program for Centers of Excellence in R&D (2017–2021) from the Agencia Estatal de Investigación of Spain (grant SEV-2016-0672 to the Centro de Biotecnología y Genómica de Plantas). I.G.-C. was supported by contract PEJ-2018-TL/BIO-11747 from Comunidad de Madrid, Spain. This study was completely supported financially by VIROPLANT, a project that received funding from the European Union’s Horizon 2020 Research and Innovation Program (grant agreement number 773567)