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Functional genomics tools to decipher the pathogenicity mechanisms of the necrotrophic fungus Plectosphaerella cucumerina in Arabidopsis thaliana.

ABSTRACT: The analysis of the interaction between Arabidopsis thaliana and adapted (PcBMM) and nonadapted (Pc2127) isolates of the necrotrophic fungus Plectosphaerella cucumerina has contributed to the identification of molecular mechanisms controlling plant resistance to necrotrophs. To characterize the pathogenicity bases of the virulence of necrotrophic fungi in Arabidopsis, we developed P.?cucumerina functional genomics tools using Agrobacterium tumefaciens-mediated transformation. We generated PcBMM-GFP and Pc2127-GFP transformants constitutively expressing the green fluorescence protein (GFP), and a collection of random T-DNA insertional PcBMM transformants. Confocal microscopy analyses of the initial stages of PcBMM-GFP infection revealed that this pathogen, like other necrotrophic fungi, does not form an appressorium or penetrate into plant cells, but causes successive degradation of leaf cell layers. By comparing the colonization of Arabidopsis wild-type plants and hypersusceptible (agb1-1 and cyp79B2cyp79B3) and resistant (irx1-6) mutants by PcBMM-GFP or Pc2127-GFP, we found that the plant immune response was already mounted at 12-18 h post-inoculation, and that Arabidopsis resistance to these fungi correlated with the time course of spore germination and hyphal growth on the leaf surface. The virulence of a subset of the PcBMM T-DNA insertional transformants was determined in Arabidopsis wild-type plants and agb1-1 mutant, and several transformants were identified that showed altered virulence in these genotypes in comparison with that of untransformed PcBMM. The T-DNA flanking regions in these fungal mutants were successfully sequenced, further supporting the utility of these functional genomics tools in the molecular characterization of the pathogenicity of necrotrophic fungi.

SUBMITTER: Ramos B

PROVIDER: S-EPMC6638842 | biostudies-literature | 2013 Jan

REPOSITORIES: biostudies-literature

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Functional genomics tools to decipher the pathogenicity mechanisms of the necrotrophic fungus Plectosphaerella cucumerina in Arabidopsis thaliana.

Ramos Brisa B González-Melendi Pablo P Sánchez-Vallet Andrea A Sánchez-Rodríguez Clara C Sánchez-Rodríguez Clara C López Gemma G Molina Antonio A

Molecular plant pathology 20120903 1

The analysis of the interaction between Arabidopsis thaliana and adapted (PcBMM) and nonadapted (Pc2127) isolates of the necrotrophic fungus Plectosphaerella cucumerina has contributed to the identification of molecular mechanisms controlling plant resistance to necrotrophs. To characterize the pathogenicity bases of the virulence of necrotrophic fungi in Arabidopsis, we developed P. cucumerina functional genomics tools using Agrobacterium tumefaciens-mediated transformation. We generated PcBMM- ...[more]

PMID: 22937870

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Project description:BackgroundXylanase can replace chemical additives to improve the volume and sensory properties of bread in the baking. Suitable baking xylanase with improved yield will promote the application of xylanase in baking industry. The xylanase XYNZG from the Plectosphaerella cucumerina has been previously characterized by heterologous expression in Pichia pastoris. However, P. pastoris is not a suitable host for xylanase to be used in the baking process since P. pastoris does not have GRAS (Generally Regarded As Safe) status and requires large methanol supplement during the fermentation in most conditions, which is not allowed to be used in the food industry. Kluyveromyces lactis, as another yeast expression host, has a GRAS status, which has been successfully used in food and feed applications. No previous work has been reported concerning the heterologous expression of xylanase gene xynZG in K. lactis with an aim for application in baking.ResultsThe xylanase gene xynZG from the P. cucumerina was heterologously expressed in K. lactis. The recombinant protein XYNZG in K. lactis presented an approximately 19 kDa band on SDS-PAGE and zymograms analysis. Transformant with the highest halo on the plate containing the RBB-xylan (Remazol Brilliant Blue-xylan) was selected for the flask fermentation in different media. The results indicated that the highest activity of 115 U/ml at 72 h was obtained with the YLPU medium. The mass spectrometry analysis suggested that the hydrolytic products of xylan by XYNZG were mainly xylobiose and xylotriose. The results of baking trials indicated that the addition of XYNZG could reduce the kneading time of dough, increase the volume of bread, improve the texture, and have more positive effects on the sensory properties of bread.ConclusionsXylanase XYNZG is successfully expressed in K. lactis, which exhibits the highest activity among the published reports of the xylanase expression in K. lactis. The recombinant XYNZG can be used to improve the volume and sensory properties of bread. Therefore, the expression yield of recombinant XYNZG can be further improved through engineered strain containing high copy numbers of the XYNZG, and optimized fermentation condition, making bread-baking application possible.

Dataset Information

Functional genomics tools to decipher the pathogenicity mechanisms of the necrotrophic fungus Plectosphaerella cucumerina in Arabidopsis thaliana.

Publications

Functional genomics tools to decipher the pathogenicity mechanisms of the necrotrophic fungus Plectosphaerella cucumerina in Arabidopsis thaliana.

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