Insight into the molecular requirements for pathogenicity of Fusarium oxysporum f. sp. lycopersici through large-scale insertional mutagenesis

An insertional mutagenesis screen identifies pathogenicity-related genes in the plant fungal pathogen Fusarium oxysporum

The Nuclear Protein Sge1 of Fusarium oxysporum Is Required for Parasitic Growth

Dimorphism or morphogenic conversion is exploited by several pathogenic fungi and is required for tissue invasion and/or survival in the host. We have identified a homolog of a master regulator of this morphological switch in the plant pathogenic fungus Fusarium oxysporum f. sp. lycopersici. This non-dimorphic fungus causes vascular wilt disease in tomato by penetrating the plant roots and colonizing the vascular tissue. Gene knock-out and complementation studies established that the gene for this putative regulator, SGE1 (SIX Gene Expression 1), is essential for pathogenicity. In addition, microscopic analysis using fluorescent proteins revealed that Sge1 is localized in the nucleus, is not required for root colonization and penetration, but is required for parasitic growth. Furthermore, Sge1 is required for expression of genes encoding effectors that are secreted during infection. We propose that Sge1 is required in F. oxysporum and other non-dimorphic (plant) pathogenic fungi for parasitic growth

Intragenic deletion in the LARGE gene causes Walker-Warburg syndrome

Intragenic homozygous deletions in the Large gene are associated with a severe neuromuscular phenotype in the myodystrophy (myd) mouse. These mutations result in a virtual lack of glycosylation of α-dystroglycan. Compound heterozygous LARGE mutations have been reported in a single human patient, manifesting with mild congenital muscular dystrophy (CMD) and severe mental retardation. These mutations are likely to retain some residual LARGE glycosyltransferase activity as indicated by residual α-dystroglycan glycosylation in patient cells. We hypothesized that more severe LARGE mutations are associated with a more severe CMD phenotype in humans. Here we report a 63-kb intragenic LARGE deletion in a family with Walker-Warburg syndrome (WWS), which is characterized by CMD, and severe structural brain and eye malformations. This finding demonstrates that LARGE gene mutations can give rise to a wide clinical spectrum, similar as for other genes that have a role in the post-translational modification of the α-dystroglycan protein

Agrobacterium-Mediated Transformation of Aspergillus awamori in the Absence of Full-Length VirD2, VirC2, or VirE2 Leads to Insertion of Aberrant T-DNA Structures

Reductions to 2, 5, and 42% of the wild-type transformation efficiency were found when Agrobacterium mutants carrying transposon insertions in virD2, virC2, and virE2, respectively, were used to transform Aspergillus awamori. The structures of the T-DNAs integrated into the host genome by these mutants were analyzed by Southern and sequence analyses. The T-DNAs of transformants obtained with the virE2 mutant had left-border truncations, whereas those obtained with the virD2 mutant had truncated right ends. From this analysis, it was concluded that the virulence proteins VirD2 and VirE2 are required for full-length T-DNA integration and that these proteins play a role in protecting the right and left T-DNA borders, respectively. Multicopy and truncated T-DNA structures were detected in the majority of the transformants obtained with the virC2 mutant, indicating that VirC2 plays a role in correct T-DNA processing and is required for single-copy T-DNA integration

Comparative transcriptome and proteome analysis reveals a global impact of the nitrogen regulators AreA and AreB on secondary metabolism in Fusarium fujikuroi.

The biosynthesis of multiple secondary metabolites in the phytopathogenic ascomycete Fusarium fujikuroi is strongly affected by nitrogen availability. Here, we present the first genome-wide transcriptome and proteome analysis that compared the wild type and deletion mutants of the two major nitrogen regulators AreA and AreB. We show that AreB acts not simply as an antagonist of AreA counteracting the expression of AreA target genes as suggested based on the yeast model. Both GATA transcription factors affect a large and diverse set of common as well as specific target genes and proteins, acting as activators and repressors. We demonstrate that AreA and AreB are not only involved in fungal nitrogen metabolism, but also in the control of several complex cellular processes like carbon metabolism, transport and secondary metabolism. We show that both GATA transcription factors can be considered as master regulators of secondary metabolism as they affect the expression of more than half of the 47 putative secondary metabolite clusters identified in the genome of F. fujikuroi. While AreA acts as a positive regulator of many clusters under nitrogen-limiting conditions, AreB is able to activate and repress gene clusters (e.g. bikaverin) under nitrogen limitation and sufficiency. In addition, ChIP analyses revealed that loss of AreA or AreB causes histone modifications at some of the regulated gene clusters

Overview of the main AreA- and AreB-mediated regulations of secondary metabolite cluster expression at nitrogen-limiting (6 mM glutamine) and nitrogen-sufficient (60 mM glutamine) conditions.

<p>Blue arrows indicate positive regulations, red lines indicate repressing effects. Black arrows indicate expression of clusters in the Wt under the respective nitrogen condition. (A) Clusters activated by AreA only; (B) clusters synergistically activated by AreA and AreB; (C) clusters repressed by AreB only; (D) clusters activated by AreB only.</p

H3K9 acetylation is affected in Δ<i>AREA</i> and Δ<i>AREB</i> at the GA and FUB gene cluster.

<p>The <i>F</i>. <i>fujikuroi</i> Wt and the Δ<i>AREA</i> and Δ<i>AREB</i> deletion mutants were cultivated for 3 days in ICI liquid cultures with 6 mM (A, B, D) or 60 mM (C) glutamine as sole nitrogen source. The mycelium was subsequently cross-linked and used for chromatin immunoprecipitation (ChIP) experiments using an anti-H3K9ac antibody (AM39137). The precipitated amount of DNA was quantified at (A) GA (<i>P450-1</i> and <i>P450-2</i>), (B) FUM (<i>FUM1</i> and <i>FUM8</i>), (C) FA (<i>FUB1</i> and <i>FUB5</i>) and (D) BIK (<i>BIK1</i> and <i>BIK2</i>) cluster genes by qPCR. In each case, the amount of DNA in the Wt was arbitrarily set to 1. Mean values and standard deviations are shown. Experiments were done in technical and biological replicates.</p

Selection of differentially regulated transcription factor genes in Δ<i>AREA</i> and Δ<i>AREB</i> cultivated with 6 mM glutamine and 60 mM glutamine.

<p>Selection of differentially regulated transcription factor genes in Δ<i>AREA</i> and Δ<i>AREB</i> cultivated with 6 mM glutamine and 60 mM glutamine.</p