Explore the vast range of titles available.

The Ascomycetous fungus Bipolaris oryzae is the causal agent of brown leaf spot disease in rice and is a model for studying photomorphogenetic responses by near-UV radiation. Targeted gene disruption (knockout) for functional analysis of photomorphogenesis-related genes in B. oryzae can be achieved by homologous recombination with low efficiency. Here, the applicability of RNA silencing (knockdown) as a tool for targeting endogenous genes in B. oryzae is reported. A polyketide synthase gene (PKS1), involved in fungal DHN melanin biosynthesis pathways, was targeted by gene silencing as a marker. The silencing vector encoding hairpin RNA of the PKS1 fragment was constructed in a two-step PCR-based cloning, and introduced into the B. oryzae genomic DNA. Silencing of the PKS1 gene resulted in albino phenotypes and reduction of PKS1 mRNA expression. These results demonstrate the applicability of targeted gene silencing as a useful reverse-genetics approach in B. oryzae.

The melanin produced by Aureobasidium melanogenum XJ5-1 obtained from the Taklimakan Desert can play an important role in adaptation of the yeast strain to various stress treatments. It is very important to know how the desert-derived yeast sense, respond and adapt to the harsh environments. However, it is still unclear how melanin is genetically controlled by signaling pathways and transcriptional factors. In this study, it was found that the mitogen-activated protein kinase (MAPK) Slt2 in the cell wall integrity (CWI) signal pathway could regulate activity of the transcriptional activator Swi4; in turn, the Swi4 could control the expression of the CMR1 gene. The melanin-specific transcriptional activator Cmr1 encoded by the CMR1 gene was specifically bound to the promoter with the sequence TTCTCTCCA of the PKS1 gene and strongly stimulated expression of the PKS1 gene and any other genes responsible for melanin biosynthesis, so that a large amount of melanin could be produced by A. melanogenum XJ5-1. Therefore, melanin biosynthesis in the desert-derived A. melanogenum XJ5-1 was controlled mainly by the CWI signal pathway among the cell wall-related signal pathways via a transcriptional activator Cmr and regulation of the melanin biosynthesis in A. melanogenum XJ5-1 was completely different from that of the melanin biosynthesis in any other fungi. This is the first time to show that melanin biosynthesis in the desert-derived A. melanogenum XJ5-1 is controlled mainly by the CWI signal pathway via a transcriptional activator Cmr1. This would provide the fundamentals for further research on the desert-derived yeast to sense, respond and adapt to the harsh environments.

Background Members of the fungal genus Trichoderma are well-known for their mycoparasitic and plant protecting activities, rendering them important biocontrol agents. One of the most significant specialized metabolites (SMs) produced by various Trichoderma species is the unsaturated lactone 6-pentyl-alpha-pyrone (6-PP). Although first identified more than 50 years ago and having pronounced antifungal and plant growth-promoting properties, the biosynthetic pathway of 6-PP still remains unresolved. Results Here, we demonstrate that 6-PP is biosynthesized via the polyketide biosynthesis pathway. We identified Pks1, an iterative type I polyketide synthase, as crucial for its biosynthesis in Trichoderma atroviride , a species recognized for its prominent 6-PP production abilities. Phylogenetic and comparative genomic analyses revealed that the pks1 gene is part of a biosynthetic gene cluster conserved in those Trichoderma species that are known to produce 6-PP. Deletion of pks1 caused a complete loss of 6-PP production in T. atroviride and a significant reduction in antifungal activity against Botrytis cinerea and Rhizoctonia solani . Surprisingly, the absence of pks1 led to enhanced lateral root formation in Arabidopsis thaliana during interaction with T. atroviride . Transcriptomic analysis revealed co-regulation of pks1 with adjacent genes, including candidates coding for a C3H1-type zinc finger protein and lytic polysaccharide monooxygenase, suggesting coordination between 6-PP biosynthesis and environmental response mechanisms. Conclusion Our findings establish pks1 as an essential gene for 6-PP biosynthesis in T. atroviride , providing novel insights into the production of one of the most significant compounds of this mycoparasite. These findings may pave the way for the development of improved biocontrol agents and the application of 6-PP as potent biopesticide contributing to an eco-friendly and sustainable way of plant disease management.

Red rot is one of the major threatening diseases of sugarcane. Due to frequent breakdown of resistance by simultaneous evolution of pathogen variants with the introduction of newer varieties red rot remains as a challenge for successful breeding programme in India. Earlier studies established that the pathogen has limited genotypic variation with enormous phenotypic variation, which could be mainly due to adaptability of pathogen to the newly released varieties and expression of pathogenicity factors including secondary metabolites viz., toxins, enzymes and pigments like melanin. It is also proved that a positive correlation remains between the production of melanin and disease expression and hence the melanin biosynthesizing genes viz., Polyketide synthase1 (PKS1), Scytalone dehydratase1 (SCD1) and Trihydroxynapthalene reductase1 (THR1) were characterized. In this study, we have utilized RNA interference (RNAi) approach using pSilent-1 vector which facilitates the generation of hair pin constructs that suppress the expression of target gene through Agrobacterium mediated transformation (ATMT) to functionally analyze the major gene PKS1 involved in the production of dihydroxy naphthalene (DHN) melanin to determine its role in virulence in C. falcatum. Functionally active sites of the PKS1 gene i.e., acyl transferase (AT) domain and the region responsible for conidial pigment (CP) polyketide synthase were chosen for knockdown approach. The above said regions were amplified in the virulent C. falcatum isolate (Cf671) and both the sense and antisense fragments were separately cloned into pSilent-1 vector. The expression cassette was then cloned into the binary vector and introduced into Agrobacterium tumefaciens LBA4404 which was later co-cultivated with the fungal spore suspension. Results indicated that the knockdown AT mutant failed to produce spores whereas the CP mutants produced spores as that of the parental strains. Further pathogenicity testing revealed that the disease symptoms of both AT and CP mutants were restricted within the inoculated internode until 15th and 5th dpi respectively while the symptoms of wild type were prominent and crossed beyond 3rd internode at 15 dpi during the host-pathogen interaction. Molecular confirmation of the knockdown mutants with the expression of hygromycin gene and absence of functional domains under RT-PCR clearly indicated that the PKS1 gene has a possible role in C. falcatum pathogenesis. However expression analysis by qRT-PCR indicated regaining of virulence in mutants during later part of host pathogen interaction. The results confirm the applicability of RNAi mediated silencing for functional analysis of pathogenicity gene homologs in C. falcatum.

Ascochyta rabiei produces and accumulates one of the well-known fungal polyketides, 1,8-dihydroxynaphthalene-melanin pigment (DHN-melanin), in asexual and sexual fruiting bodies. Degenerate PCR primers were used to isolate an ArPKS1 of A. rabiei encoding a polypeptide with high similarity to polyketide synthase (PKS) involved in biosynthesis of DHN-melanin in other ascomycetous fungi. Site-directed mutagenesis of ArPKS1 in A. rabiei generated melanin-deficient pycnidial mutants but caused no significant reduction of pathogenicity to chickpea. Pycnidiospores in ArPKS1 -mutant pycnidia showed higher sensitivity to UV light exposure compared to pycnidiospores in melanized pycnidia of the wild-type progenitor isolate. Integration of an orthologous PKS1 gene from Bipolaris oryzae into the genome of the mutants complemented the dysfunctional ArPKS1 gene. This study demonstrated that A. rabiei uses a DHN-melanin pathway for pigmentation of pycnidia and this molecule may protect pycnidiospores from UV irradiation.

Phytochrome kinase substrate1 (PKS1) is a cytoplasmic protein that interacts physically with, and is phosphorylated by, the plant photoreceptor phytochrome. Here, we show that light transiently increases PKS1 mRNA levels and concentrates its expression to the elongation zone of the hypocotyl and root. This response is mediated by phytochrome A (phyA) acting in the very low fluence response (VLFR) mode. In the hypocotyl, PKS1 RNA and protein accumulation are maintained only under prolonged incubation in far-red light, the wavelength that most effectively activates phyA. Null mutants of PKS1 and its closest homolog, PKS2, show enhanced phyA-mediated VLFR. Notably, a pks1 pks2 double mutant has no phenotype, whereas overexpression of either PKS1 or PKS2 results in the same phenotype as the pks1 or pks2 single null mutant. We propose that PKS1 and PKS2 are involved in a growth regulatory loop that provides homeostasis to phyA signaling in the VLFR. In accordance with this idea, PKS1 effects are larger in the pks2 background (and vice versa). Moreover, the two proteins can interact with each other, and PKS2 negatively regulates PKS1 protein levels specifically under VLFR conditions.

A polyketide synthase gene named PKS1, involved in the melanin biosynthesis pathway of the phytopathogenic fungus Bipolaris oryzae, was isolated using restriction enzyme-mediated integration. Sequence analysis showed that the PKS1 encodes a putative protein that has 2155 amino acids and significant similarity to other fungal polyketide synthases. Targeted disruption of the PKS1 gene showed that it is necessary for melanin biosynthesis in B. oryzae. Northern blot analysis showed that PKS1 transcripts were specifically enhanced by near-ultraviolet radiation (300–400 nm) and that its temporal transcriptional patterns were similar to those of THR1 and SCD1 genes involved in the melanin biosynthesis pathway of B. oryzae.

Three polyketide synthase genes (PKS1, PKS2, PKS3) from cell suspension cultures of raspberry (Rubus idaeus L. cv. Royalty) were characterized. They showed high similarity in both their nucleotide and deduced amino acid sequences. All three proteins contain the amino acid residues identified in previous work as essential for chalcone synthase (CHS) function. Enzyme activities were investigated after heterologous expression in Escherichia coli. RiPKS1 is a typical naringenin CHS that synthesizes the chalcone as the main reaction product, and p-coumaryltriacetic acid lactone (CTAL) as a minor by-product. RiPKS3 differed from RiPKS1 in four positions (K49R, M64R, P120L, V188A), and the products in vitro were predominantly CTAL and low levels of chalcone. RiPKS2 had the same four differences from RiPKS1 as RiPKS3, but in addition two further exchanges (R259H, F344L), and the protein had no detectable enzyme activity. Experiments with RiPKS1 containing either 259H or 344L showed that each of the exchanges was sufficient to completely eliminate enzyme activity. These experiments identify amino acid residues in CHS which are important for folding of the tetraketide intermediate to the chalcone (PKS3) and which are in general essential for CHS activity (PKS2). The possible functions of these residues are discussed.

Abstract A polyketide synthase gene named PKS1, involved in the melanin biosynthesis pathway of the phytopathogenic fungus Bipolaris oryzae, was isolated using restriction enzyme-mediated integration. Sequence analysis showed that the PKS1 encodes a putative protein that has 2155 amino acids and significant similarity to other fungal polyketide synthases. Targeted disruption of the PKS1 gene showed that it is necessary for melanin biosynthesis in B. oryzae. Northern blot analysis showed that PKS1 transcripts were specifically enhanced by near-ultraviolet radiation (300–400 nm) and that its temporal transcriptional patterns were similar to those of THR1 and SCD1 genes involved in the melanin biosynthesis pathway of B. oryzae.

We characterized a spontaneous albino mutant of Ceratocystis resinifera. Compared with the wild-type progenitor strain, the albino mutant had a reduced linear growth on culture medium, but its growth on lodgepole pine sapwood was unaffected. The albino mutant did not produce any coloured pigment on agar media or wood. However, upon exposure to exogenous scytalone, an intermediate metabolite of the melanin pathway, the production of a brownish melanin was restored. This suggests that the albino phenotype resulted from a mutation affecting the melanin synthesis pathway, upstream of the scytalone synthesis step. Melanin production was restored in the mutant by transforming it with a wild-type copy of the Ceratocystis resinifera polyketide synthase gene, PKS1. The complemented transformants produced melanin, indicating that the PKS1 gene was defective in the albino mutant. Sequence analysis revealed that the PKS1 allele found in the albino contained a single point mutation that resulted in an amino acid change from serine to proline at the 3′ end of the β-ketoacyl synthase motif.Key words: Ceratocystis resinifera, polyketide synthase, sap stain, albino mutant, biological control.