1.8.13
IDENTIFICATION OF DETERMINANTS ESSENTIAL FOR COLLETOTRICHUM LINDEMUTHIANUM PATHOGENICITY ON COMMON BEAN BY INSERTIONAL MUTAGENESIS

T LANGIN, M DUFRESNE and D PARISOT

CNRS, Orsay cedex, France

Background and objectives
Following an insertional mutagenesis strategy based on the integration of the pAN7-1 vector, nine Colletotrichum lindemuthianum mutant strains, more or less affected in their pathogenicity on a susceptible cultivar of common bean, were obtained out of 1100 transformants.

Results and conclusions
One of these strains, the H433 mutant strain, is characterized by the absence of normal anthracnose symptoms and the ability to induce local necrotic spots similar to those observed in a classical hypersentive response (HR). Cytological analyses revealed slight differences with the HR. In particular, the H433 strain is able to differentiate primary hyphae but not secondary hyphae. Plasmolysis experiments revealed that the H433 strain appeared to be able to establish a normal biotrophic phase, but that cells infected by the mutant started to die during the growth of primary hyphae. Therefore, this mutant strain appears to be blocked at a critical step of the infection process, the transition between biotrophy and necrotrophy. The expression of genes involved in defence reactions are analysed by both RT-PCR experiments and Northern blot analysis. The mutant results from the insertion of pAN7-1 copies at two independant loci in the C. lindemuthianum recipient strain genome. The two corresponding wild-type genomic regions, called 433.1 and 433.2, were used to complement the H433 mutant strain. The results revealed that only 433.2 was able to restore partially a wild-type phenotype. The complementing region was contained within a 4.0-kbp HindIII fragment. Sequencing of this fragment identified a single 1785-bp open reading frame, encoding a putative polypeptide of 594 amino acids sharing significant homologies with transcriptional activators of the GAL4 family. The gene was called clta1 (for C. lindemuthianum transcriptional activator 1). The involvment of the clta1 gene in C. lindemuthianum pathogenicity was confirmed by gene replacement experiments. Transformants in which the wild-type allele was replaced by the mutant allele constructed in vitro shared phenotypes with the original mutant strain H433: inability to induce the appearance of anthracnose symptoms, and the induction of necrotic spots.

References
1. Dufresne M, Parisot D, Dron M, Langin T, 1998. Molecular Plant-Microbe Interactions (in press).