Construction of gateway binary vector for selection with bialaphos or carboxin and GFP expression in fungi.

Genomic data has created a growing demand for tools and methodologies for studying the genes function, which can be realized through loss of function experiments (gene knockout) or by RNA silencing (knockdown). The develop-ment of binary vectors for Agrobacterium tumefaciens mediated transformation (ATMT) has the advantage of being independent of protoplast formation and can be used directly on a wide variety of fungal species and tissue types. The selection of transformants using bialaphos and carboxin has the advantages of low cost in the transformation and availability of different selectable markers, also allowing the analysis of several genes and combination of study by knockout or knockdown, using selectable markers in the same transformant. Thus, this study aimed to build two binary vectors containing reporter gene and selectable markers that confer resistance to carboxin and bialaphos. The cassettes were constructed using the Gateway system to two fragments. The gene encoding the GFP protein and PtoxA and PtrpCpromoters were cloned into pDONR P1-P5R plasmid. Genes that confer bialaphos and carboxin resistance, bar and cbxr respectively, were cloned into pDONR P5-P2 plasmid. The pPGW plasmid was used as des-tination vector. The gfp gene transcription is controlled by PtoxA promoter and the bar and cbxr genes transcriptions are controlled by PtrpC promoter. These binary vectors were named pGWGFP-BAR and pGWGFP-CBXR. The assembly of cassettes was confi rmed by sequencing, and the validation of vectors is being accomplished through transformation (ATMT) with the plant pathogens Mycosphaerella fi jiensis and Fusarium oxysporum f. sp. cubense

Triazole Fungicides Can Induce Cross-Resistance to Medical Triazoles in Aspergillus fumigatus

Contains fulltext : 103858.pdf (publisher's version ) (Open Access)BACKGROUND: Azoles play an important role in the management of Aspergillus diseases. Azole resistance is an emerging global problem in Aspergillus fumigatus, and may develop through patient therapy. In addition, an environmental route of resistance development has been suggested through exposure to 14alpha-demethylase inhibitors (DMIs). The main resistance mechanism associated with this putative fungicide-driven route is a combination of alterations in the Cyp51A-gene (TR(34)/L98H). We investigated if TR(34)/L98H could have developed through exposure to DMIs. METHODS AND FINDINGS: Thirty-one compounds that have been authorized for use as fungicides, herbicides, herbicide safeners and plant growth regulators in The Netherlands between 1970 and 2005, were investigated for cross-resistance to medical triazoles. Furthermore, CYP51-protein homology modeling and molecule alignment studies were performed to identify similarity in molecule structure and docking modes. Five triazole DMIs, propiconazole, bromuconazole, tebuconazole, epoxiconazole and difenoconazole, showed very similar molecule structures to the medical triazoles and adopted similar poses while docking the protein. These DMIs also showed the greatest cross-resistance and, importantly, were authorized for use between 1990 and 1996, directly preceding the recovery of the first clinical TR(34)/L98H isolate in 1998. Through microsatellite genotyping of TR(34)/L98H isolates we were able to calculate that the first isolate would have arisen in 1997, confirming the results of the abovementioned experiments. Finally, we performed induction experiments to investigate if TR(34)/L98H could be induced under laboratory conditions. One isolate evolved from two copies of the tandem repeat to three, indicating that fungicide pressure can indeed result in these genomic changes. CONCLUSIONS: Our findings support a fungicide-driven route of TR(34)/L98H development in A. fumigatus. Similar molecule structure characteristics of five triazole DMIs and the three medical triazoles appear the underlying mechanism of cross resistance development. Our findings have major implications for the assessment of health risks associated with the use of triazole DMIs

Genetic analysis of resistance to septoria tritici blotch in the French winter wheat cultivars Balance and Apache

The ascomycete Mycosphaerella graminicola is the causal agent of septoria tritici blotch (STB), one of the most destructive foliar diseases of bread and durum wheat globally, particularly in temperate humid areas. A screening of the French bread wheat cultivars Apache and Balance with 30 M. graminicola isolates revealed a pattern of resistant responses that suggested the presence of new genes for STB resistance. Quantitative trait loci (QTL) analysis of a doubled haploid (DH) population with five M. graminicola isolates in the seedling stage identified four QTLs on chromosomes 3AS, 1BS, 6DS and 7DS, and occasionally on 7DL. The QTL on chromosome 6DS flanked by SSR markers Xgpw5176 and Xgpw3087 is a novel QTL that now can be designated as Stb18. The QTLs on chromosomes 3AS and 1BS most likely represent Stb6 and Stb11, respectively, and the QTLs on chromosome 7DS are most probably identical with Stb4 and Stb5. However, the QTL identified on chromosome 7DL is expected to be a new Stb gene that still needs further characterization. Multiple isolates were used and show that not all isolates identify all QTLs, which clearly demonstrates the specificity in the M. graminicola–wheat pathosystem. QTL analyses were performed with various disease parameters. The development of asexual fructifications (pycnidia) in the characteristic necrotic blotches of STB, designated as parameter P, identified the maximum number of QTLs. All other parameters identified fewer but not different QTLs. The segregation of multiple QTLs in the Apache/Balance DH population enabled the identification of DH lines with single QTLs and multiple QTL combinations. Analyses of the marker data of these DH lines clearly demonstrated the positive effect of pyramiding QTLs to broaden resistance spectra as well as epistatic and additive interactions between these QTLs. Phenotyping of the Apache/Balance DH population in the field confirmed the presence of the QTLs that were identified in the seedling stage, but Stb18 was inconsistently expressed and might be particularly effective in young plants. In contrast, an additional QTL for STB resistance was identified on chromosome 2DS that is exclusively and consistently expressed in mature plants over locations and time, but it was also strongly related with earliness, tallness as well as resistance to Fusarium head blight. Although to date no Stb gene has been reported on chromosome 2D, the data provide evidence that this QTL is only indirectly related to STB resistance. This study shows that detailed genetic analysis of contemporary commercial bread wheat cultivars can unveil novel Stb genes that can be readily applied in marker-assisted breeding programs

Genetic transformation of Fusarium oxysporum f. sp. cubense.

Edição dos Resumos do 42° Congresso Brasileiro de Fitopatologia, Rio de Janeiro, ago. 2009

Developing tools for Mycosphaerella fijiensis studies.

Mycosphaerella fijiensis is the causal agent of the black leaf streak disease, or Black Sigatoka, the most devasting fungal disease of banana around the world.Edição do Fast Firward 40 jaar Gewasbescherming, 2009, Wageningen

GFP and RFP transformation of Fusarium guttiforme (sin. Fusarium subglutinans f. sp. annanas).

Edição dos Resumos do 42° Congresso Brasileiro de Fitopatologia, Rio de Janeiro, ago. 2009