Efficient gene replacement and direct hyphal transformation in Sclerotinia sclerotiorum.

Abstract:

:Homologous recombination is required for gene-targeted procedures such as gene disruption and gene replacement. Ku80 is part of the non-homologous end-joining DNA repair mechanism in many organisms. We identified and disrupted the Ku80 homologue in Sclerotinia sclerotiorum and generated heterokaryon mutants enriched with Ku80-deficient nuclei (ssku80). Sclerotial formation and pathogenicity of ssku80 mutants were normal on tomato fruits. The frequencies of homologous recombination in these strains were much higher than those of the wild type when transformed with a cna1 (encoding calcineurin) replacement construct. We coupled the increase in homologous recombination with a direct BIM-LAB-mediated transformation procedure, which utilizes compressed air to assist the transforming DNA in penetrating fungal hyphae of S. sclerotiorum. We found this method to be efficient and reproducible, and it did not alter the fitness of the mutants. We also demonstrated the first case of direct transformation of sclerotia. Nourseothricin was introduced as a selectable marker in S. sclerotiorum. The tools and procedures described will improve our ability to study gene function in S. sclerotiorum and are most likely to be adaptable for use in other plant pathogens.

journal_name

Mol Plant Pathol

authors

Levy M,Erental A,Yarden O

doi

10.1111/j.1364-3703.2008.00483.x

subject

Has Abstract

pub_date

2008-09-01 00:00:00

pages

719-25

issue

5

eissn

1464-6722

issn

1364-3703

pii

MPP483

journal_volume

9

pub_type

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