Determination of fungal gene expression in planta by qRT-PCR and characterization of putative pathogenicity related genes of Verticillium longisporum
by Hai Quan Xu
Date of Examination:2011-02-16
Date of issue:2013-02-26
Advisor:Prof. Dr. Petr Karlovsky
Referee:Prof. Dr. Andreas von Tiedemann
Referee:Prof. Dr. Petr Karlovsky
Files in this item
Name:Xu-Manuscript 1V.pdf
Size:1.80Mb
Format:PDF
Abstract
English
Genes of Verticillium longisporum potentially involved in the pathogenicity of the fungus were identified in previous studies. The purpose of this work was to characterize some of these genes. Short transcript-derived fragment obtained after differential cDNA-AFLP were used as the starting material. Full-length sequences were obtained using RACE-PCR (rapid amplification of cDNA-ends with polymerase chain reaction), inverse PCR on self circularized genomic DNA, by screening of a genomic library and by cloning PCR products generated with primers based on published sequences from the genome of V. dahliae. A genomic library of Verticillium longisporum was constructed, consisting of about 10,000 plasmids arrayed in 96-wells microplates. The insert size was 8-12 kb. The library represented approximately 50% of the genome. The library was hierarchically ordered and pools of whole microplates, rows and columns were generated. These pools allowed the identification of a single sequence in the library by PCR with less than 40 PCR reactions. Full-length genomic copies of several candidate genes were obtained in this way. Analysis of the expression of candidate genes in planta by real-time RT-PCR was established. Fungus colonizing the root and shoot tissue of infected Brassica napus plants was analyzed separately. The isolation, purification and transcription procedures of total RNA and mRNA from V. longisporum infected Brassica plants were improved and used for the determination of relative gene expression by qRT-PCR. A set of reference genes were tested. The in planta determination of relative gene expression of pathogenicity related genes in V. longisporum compared to growth in vitro in a xylem-simulating medium (SXM) by quantitative reverse transcription real-time PCR (qRT-PCR) was applied for description of TDFs after cDNA-AFLP screening and sequence extensions. 10 genes have been characterised after their relative expression levels in planta by qRT-PCR, 6 of them indicated an up-regulated gene expression, 2 of them were down-regulated and further 2 were time-depended. One of the candidate genes, designated as Vl_6.2, was identified in previous work due to strong induction of its expression by xylem sap metabolites of B. napus. This finding was confirmed by transcript analysis in planta. Vl_12.1 gene, its gene expression was reduced during infection of B. napus in root/hypocotyl tissue in reference to in vitro grown mycelium in a xylem simulating artificial medium, indicating a suppression of this gene during the infection process. The gene has an ORF of 2,328 nucleotides, one intron and translated cDNA is predicted to code for a 775 amino acids. Sequence analysis of Vl_12.1 showed the high homology to the zinc-finger transcription factor ACE1 containing three ZnF_C2H2 domains. We applied RNAi technology for gene silencing of this ACE1-like Vl_12.1 gene in V. longisporum by expressing a gene-specific RNA-hairpin. Gene-silenced mutants did not show any visual difference in triggering typical infection symptoms in B. napus. A semi-quantitative testing for alterations in cellulase activities on cellulose-containing agar medium showed less difference between the silenced mutants and V. longisporum wild type.
Keywords: Verticillium longisporum, qRT-PCR, fungal gene expression, pathogenicity, GFP