Elucidation of chemical interactions of Verticillium longisporum with Brassica napus

• Applicant: Professor Dr. Petr Karlovsky
• Subject Area: Plant Breeding and Plant Pathology ; Organismic Interactions, Chemical Ecology and Microbiomes of Plant Systems
• Term: from 2004 to 2012
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 5471048

• Final Report Year: 2012

Final Report Abstract

Our goal was to shed light onto chemical interactions between the pathogen and its host and characterize genes of the fungus involved in the infection. In the first phase, (i) low-moleculeweight components of xylem sap affected by infection were detected by differential metabolic profiling and (ii) genes of V.l. induced by xylem sap metabolites were identified by cDNA- AFLP. Defense-related compounds and virulence factor genes were sought among these metabolites and genes, respectively. Salicylic acid (SA) and SA glycoside (SAG) were identified in xylem sap of B. napus with levels increased after infection. Relationships between SA, SAG, phytohormone levels, disease severity and fungal DNA content were studied. In order to determine the structures of further xylem metabolites, xylem sap from over 7,000 V.l.-infected B. napus plants was collected. 42-times enrichment of one of the metabolites (MW 612) by preparative HPLC was achieved with a yield of 85%. This work has not been finished, it continues past FOR546 funding period. Quantification of the expression of V. longisporum genes in planta and suppression of fungal genes by RNAi and antisense RNA were established. Both methods were applied to a set of candidate genes. Most interesting phenotypes were observed after the suppression of Vl_6.2 gene putatively involved in cellular integrity, polyketide synthase gene PKS1, and NEP-like protein-encoding gene VlNEP-1. All three genes were strongly induced in planta. Vl_62 product putatively binds carbohydrates; the silenced mutants exhibited reduced virulence and increased susceptibility to detergents. Silenced mutants of PKS1 were not impaired in virulence but exhibited increased growth rate, delayed pigmentation on agar and a loss of orange pigment in potato dextrose broth; their competitive fitness was also reduced. Strains silenced in VlNEP-1 were impaired in virulence. Purified VlNEP-1 protein exerted strong phytotoxic effects on B. napus, phytotoxicity to A. thaliana differed between V.l.-susceptible and V.l.-resistant ecotypes. Histological studies showed that the protein is released into xylem and infiltrates surrounding tissues. We hypothesize that its function is to release nutrients into xylem by permeabilizing membranes of the surrounding cells. As a service we established and provided determination of fungal biomass in plant tissue, constructed labeled V.l. strains and wrote Perl scripts for processing cDNA-AFLP data generated with B. napus.

Publications

• (2008) Defence reactions in the apoplastic proteome of oilseed rape (Brassica napus var. napus) attenuate Verticillium longisporum growth but not disease symptoms. BMC Plant Biol 8: 129
  Floerl S, Druebert C, Majcherczyk A, Karlovsky P, Kües U, Polle A
  
• (2008) Improved coverage of cDNA-AFLP by sequential digestion of immobilized cDNA. BMC Genomics 9: 480
  Weiberg A, Pöhler D, Morgenstern B, Karlovsky P
  
• (2008): Development and application of LC-MS-based differential metabolic profiling in plant systems. PhD thesis
  Astrid Ratzinger
  
• (2008): Identification of genes induced in the vascular pathogen Verticillium longisporum by xylem sap metabolites of Brassica napus using an improved genome-wide quantitative cDNA-AFLP. PhD thesis
  Arne Weiberg
  
• (2009) Components of variance in transcriptomics based on electrophoretic separation of cDNA fragments (cDNA-AFLP). Electrophoresis 30: 2549–2557
  Weiberg A, Karlovsky P
  
• (2009) Internal resistance in winter oilseed rape inhibits systemic spread of the vascular pathogen Verticillium longisporum. Phytopathology 99: 802–811
  Eynck C, Koopmann B, Karlovsky P, von Tiedemann A
  
• (2009) Salicylic acid and salicylic acid glucoside in xylem sap of Brassica napus infected with Verticillium longisporum. J Plant Res 122: 571–579
  Ratzinger A, Riediger N, von Tiedemann A, Karlovsky P
  
• (2010) Genetic and environmental control of the Verticillium syndrome in Arabidopsis thaliana. BMC Plant Biol 10: 235
  Häffner E, Karlovsky P, Diederichsen E
  
• (2010): Determination of fungal gene expression in planta by qRT-PCR and characterization of putative pathogenicity related genes of Verticillium longisporum. PhD thesis
  Haiquan Xu
  
• (2011): Molecular and functional characterization of potential pathogenicity related genes from Verticillium longisporum. PhD thesis
  Malte Beinhoff
  
• (2012) The plant host Brassica napus induces in the pathogen Verticillium longisporum the expression of functional catalase peroxidase which is required for the late phase of disease. Molecular Plant Microbe Interaction 25, 569-581
  Singh S, Braus-Stromeyer SA, Timpner C, Valerius O, von Tiedemann A, Karlovsky P, Druebert C, Polle A, Braus GH