Identification of the polyketide synthase gene responsible for the synthesis of tanzawaic acids in Penicillium steckii IBWF104-06.

Microorganisms have been used as biological control agents (BCAs) in agriculture for a long time, but their importance has increased dramatically over the last few years. The Penicillium steckii IBWF104-06 strain has presented strong BCA activity in greenhouse experiments performed against phytopathogenic fungi and oomycetes. P. steckii strains generally produce different antifungal tanzawaic acids; interesting compounds known to be catalyzed by polyketide synthetases in other fungi. Since the decalin structure is characteristic for tanzawaic acids, two polyketide synthase genes (PsPKS1 and PsPKS2) were selected for further analysis, which have similarity in sequence and gene cluster structure with genes that are known to be responsible for the biosynthesis of decalin-containing compounds. Subsequently, gene-inactivation mutants of both PsPKS1 and PsPKS2 have been generated. It was found, that the ΔPspks1 mutant cannot produce tanzawaic acids any more, whereas reintegration of the original PsPKS1 gene into the genome of ΔPspks1 reestablished tanzawaic acid production. The mutant ΔPspks2 is not altered in tanzawaic acids production. Interestingly, both mutants ΔPsPKS1 and ΔPsPKS2 still display strong BCA activity, indicating that the mechanism of action is not related to the production of tanzawaic acids.

Two Novel Dimorphism-Related Virulence Factors of Zymoseptoria tritici Identified Using Agrobacterium-Mediated Insertional Mutagenesis.

Diseases caused by dimorphic phytopathogenic and systemic dimorphic fungi have markedly increased in prevalence in the last decades, and understanding the morphogenic transition to the virulent state might yield novel means of controlling dimorphic fungi. The dimorphic fungus Z. tritici causes significant economic impact on wheat production, and yet the regulation of the dimorphic switch, a key first step in successful plant colonization, is still largely unexplored in this fungus. The fungus is amenable to suppression by fungicides at this switch point, and the identification of the factors controlling the dimorphic switch provides a potential source of novel targets to control Septoria tritici blotch (STB). Inhibition of the dimorphic switch can potentially prevent penetration and avoid any damage to the host plant. The aim of the current work was to unveil genetic determinants of the dimorphic transition in Z. tritici by using a forward genetics strategy. Using this approach, we unveiled two novel factors involved in the switch to the pathogenic state and used reverse genetics and complementation to confirm the role of the novel virulence factors and further gained insight into the role of these genes, using transcriptome analysis via RNA-Seq. The transcriptomes generated potentially contain key determinants of the dimorphic transition.

The Biocontrol Agent and Insect Pathogen Photorhabdus luminescens Interacts with Plant Roots.

The number of sustainable agriculture techniques to improve pest management and environmental safety is rising, as biological control agents are used to enhance disease resistance and abiotic stress tolerance in crops. Here, we investigated the capacity of the Photorhabdus luminescens secondary variant to react to plant root exudates and their behavior toward microorganisms in the rhizosphere. P. luminescens is known to live in symbiosis with entomopathogenic nematodes (EPNs) and to be highly pathogenic toward insects. The P. luminescens-EPN relationship has been widely studied, and this combination has been used as a biological control agent; however, not much attention has been paid to the putative lifestyle of P. luminescens in the rhizosphere. We performed transcriptome analysis to show how P. luminescens responds to plant root exudates. The analysis highlighted genes involved in chitin degradation, biofilm regulation, formation of flagella, and type VI secretion system. Furthermore, we provide evidence that P. luminescens can inhibit growth of phytopathogenic fungi. Finally, we demonstrated a specific interaction of P. luminescens with plant roots. Understanding the role and the function of this bacterium in the rhizosphere might accelerate the progress in biocontrol manipulation and elucidate the peculiar mechanisms adopted by plant growth-promoting rhizobacteria in plant root interactions.IMPORTANCE Insect-pathogenic Photorhabdus luminescens bacteria are widely used in biocontrol strategies against pests. Very little is known about the life of these bacteria in the rhizosphere. Here, we show that P. luminescens can specifically react to and interact with plant roots. Understanding the adaptation of P. luminescens in the rhizosphere is highly important for the biotechnological application of entomopathogenic bacteria and could improve future sustainable pest management in agriculture.

Rapid adaptation of signaling networks in the fungal pathogen Magnaporthe oryzae.

BACKGROUND: One fundamental question in biology is how the evolution of eukaryotic signaling networks has taken place. "Loss of function" (lof) mutants from components of the high osmolarity glycerol (HOG) signaling pathway in the filamentous fungus Magnaporthe oryzae are viable, but impaired in osmoregulation. RESULTS: After long-term cultivation upon high osmolarity, stable individuals with reestablished osmoregulation capacity arise independently from each of the mutants with inactivated HOG pathway. This phenomenon is extremely reproducible and occurs only in osmosensitive mutants related to the HOG pathway - not in other osmosensitive Magnaporthe mutants. The major compatible solute produced by these adapted strains to cope with high osmolarity is glycerol, whereas it is arabitol in the wildtype strain. Genome and transcriptome analysis resulted in candidate genes related to glycerol metabolism, perhaps responsible for an epigenetic induced reestablishment of osmoregulation, since these genes do not show structural variations within the coding or promotor sequences. CONCLUSION: This is the first report of a stable adaptation in eukaryotes by producing different metabolites and opens a door for the scientific community since the HOG pathway is worked on intensively in many eukaryotic model organisms.

Two sets of RNAi components are required for heterochromatin formation in trans triggered by truncated transgenes.

Across kingdoms, RNA interference (RNAi) has been shown to control gene expression at the transcriptional- or the post-transcriptional level. Here, we describe a mechanism which involves both aspects: truncated transgenes, which fail to produce intact mRNA, induce siRNA accumulation and silencing of homologous loci in trans in the ciliate Paramecium We show that silencing is achieved by co-transcriptional silencing, associated with repressive histone marks at the endogenous gene. This is accompanied by secondary siRNA accumulation, strictly limited to the open reading frame of the remote locus. Our data shows that in this mechanism, heterochromatic marks depend on a variety of RNAi components. These include RDR3 and PTIWI14 as well as a second set of components, which are also involved in post-transcriptional silencing: RDR2, PTIWI13, DCR1 and CID2. Our data indicates differential processing of nascent un-spliced and long, spliced transcripts thus suggesting a hitherto-unrecognized functional interaction between post-transcriptional and co-transcriptional RNAi. Both sets of RNAi components are required for efficient trans-acting RNAi at the chromatin level and our data indicates similar mechanisms contributing to genome wide regulation of gene expression by epigenetic mechanisms.

Magnaporthe oryzae effectors MoHEG13 and MoHEG16 interfere with host infection and MoHEG13 counteracts cell death caused by Magnaporthe-NLPs in tobacco.

KEY MESSAGE: Adapted pathogens are able to modulate cell responses of their hosts most likely due to the activity of secreted effector molecules thereby enabling colonisation by ostensible nonhost pathogens. It is postulated that host and nonhost pathogens of a given plant species differ in their repertoire of secreted effector molecules that are able to suppress plant resistance. We pursued the strategy of identifying novel effectors of Magnaporthe oryzae, the causal agent of blast disease, by comparing the infection process of closely related host vs. nonhost Magnaporthe species on barley (Hordeum vulgare L.). When both types of pathogen simultaneously attacked the same cell, the nonhost isolate became a successful pathogen possibly due to potent effectors secreted by the host isolate. Microarray studies led to a set of M. oryzae Hypothetical Effector Genes (MoHEGs) which were classified as Early- and LateMoHEGs according to the maximal transcript abundance during colonization of barley. Interestingly, orthologs of these MoHEGs from a nonhost pathogen were similarly regulated when investigated in a host situation, suggesting evolutionary conserved functions. Knockout mutants of MoHEG16 from the group of EarlyMoHEGs were less virulent on barley and microscopic studies revealed an attenuated transition from epidermal to mesophyll colonization. MoHEG13, a LateMoHEG, was shown to antagonize cell death induced by M. oryzae Necrosis-and ethylene-inducing-protein-1 (Nep1)-like proteins in Nicotiana benthamiana. MoHEG13 has a virulence function as a knockout mutant showed attenuated disease progression when inoculated on barley.

The role of the Tra1p transcription factor of Magnaporthe oryzae in spore adhesion and pathogenic development.

Transcription factors play a critical regulatory role in development by binding DNA and initiating alterations in gene transcription. The transcript of the putative Magnaporthe oryzae transcription factor-encoding gene TRA1 accumulates during germination and this accumulation was previously found to depend on the transcription factor Con7p. In the current work tra1⁻ mutants were generated and these strains were found to exhibit a reduced attachment, germination, appressorium formation and virulence. Adhesion to artificial and plant surfaces was affected, and FITC-labelled concanavalin A, a lectin which inhibits attachment of Magnaporthe spores, showed a reduced affinity for mutant spore tip where it normally preferentially binds. We used microarray analysis to identify Tra1p-dependent genes from two different sources: aerial structures and conidia. Mutation of 11 Tra1p-dependent genes showed that the predicted transcription factor encoding gene TDG2 is required for normal adhesion and virulence, that the genes TDG7 and TDG4 are required for normal sporulation and that TDG6 is required for wild-type levels of spore adhesion.

Identification of factors involved in dimorphism and pathogenicity of Zymoseptoria tritici.

A forward genetics approach was applied in order to investigate the molecular basis of morphological transition in the wheat pathogenic fungus Zymoseptoria tritici. Z. tritici is a dimorphic plant pathogen displaying environmentally regulated morphogenetic transition between yeast-like and hyphal growth. Considering the infection mode of Z. tritici, the switching to hyphal growth is essential for pathogenicity allowing the fungus the host invasion through natural openings like stomata. We exploited a previously developed Agrobacterium tumefaciens-mediated transformation (ATMT) to generate a mutant library by insertional mutagenesis including more than 10,000 random mutants. To identify genes involved in dimorphic switch, a plate-based screening system was established. With this approach eleven dimorphic switch deficient random mutants were recovered, ten of which exhibited a yeast-like mode of growth and one mutant predominantly growing filamentously, producing high amount of mycelium under different incubation conditions. Using genome walking approach previously established, the T-DNA integration sites were recovered and the disrupted genomic loci of corresponding mutants were identified and validated within reverse genetics approach. As prove of concept, two of the random mutants obtained were selected for further investigation using targeted gene inactivation. Both genes deduced were found to encode known factors, previously characterized in other fungi: Ssk1p being constituent of HOG pathway and Ade5,7p involved in de novo purine biosynthesis. The targeted mutant strains defective in these genes exhibit a drastically impaired virulence within infection assays on whole wheat plants. Moreover exploiting further physiological assays the predicted function for both gene products could be confirmed in concordance with conserved biological role of homologous proteins previously described in other fungal organisms.