CRISPR/Cas with ribonucleoprotein complexes and transiently selected telomere vectors allows highly efficient marker-free and multiple genome editing in Botrytis cinerea.

Thomas Leisen,Alex Wegner,Fabian Bietz,Janina Werner,Ulrich Schaffrath,David Scheuring,Gabriel Scalliet,Felix Willmund,Matthias Hahn,Andreas Mosbach

doi:10.1371/journal.ppat.1008326

Abstract

CRISPR/Cas has become the state-of-the-art technology for genetic manipulation in diverse organisms, enabling targeted genetic changes to be performed with unprecedented efficiency. Here we report on the first establishment of robust CRISPR/Cas editing in the important necrotrophic plant pathogen Botrytis cinerea based on the introduction of optimized Cas9-sgRNA ribonucleoprotein complexes (RNPs) into protoplasts. Editing yields were further improved by development of a novel strategy that combines RNP delivery with cotransformation of transiently stable vectors containing telomeres, which allowed temporary selection and convenient screening for marker-free editing events. We demonstrate that this approach provides superior editing rates compared to existing CRISPR/Cas-based methods in filamentous fungi, including the model plant pathogen Magnaporthe oryzae. Genome sequencing of edited strains revealed very few additional mutations and no evidence for RNP-mediated off-targeting. The high performance of telomere vector-mediated editing was demonstrated by random mutagenesis of codon 272 of the sdhB gene, a major determinant of resistance to succinate dehydrogenase inhibitor (SDHI) fungicides by in bulk replacement of the codon 272 with codons encoding all 20 amino acids. All exchanges were found at similar frequencies in the absence of selection but SDHI selection allowed the identification of novel amino acid substitutions which conferred differential resistance levels towards different SDHI fungicides. The increased efficiency and easy handling of RNP-based cotransformation is expected to accelerate molecular research in B. cinerea and other fungi.

Highlights

Botrytis cinerea is a plant pathogenic ascomycete which infects more than thousand species, causing gray mold disease which is responsible for over a billion dollars of losses in fruits, vegetables and flowers every year [1]
We report on the first establishment of robust clustered regularly interspaced short palindromic repeats (CRISPR)/Cas editing in the important necrotrophic plant pathogen Botrytis cinerea based on the introduction of optimized Cas9-single guide RNA (sgRNA) ribonucleoprotein complexes (RNPs) into protoplasts
We describe the establishment of the CRISPR/Cas technology for genome editing in the gray mold fungus Botrytis cinerea, one of the economically most important plant pathogens worldwide

Summary

Introduction

Botrytis cinerea is a plant pathogenic ascomycete which infects more than thousand species, causing gray mold disease which is responsible for over a billion dollars of losses in fruits, vegetables and flowers every year [1]. Since the succinate dehydrogenase inhibitor fungicides (SDHI), no new major antifungal modes of action have been released for the control of Botrytis in the last years [4]. Within this fungicidal class, new molecules are developed which display higher intrinsic activities, better physicochemical properties and reduced risk compared to existing solutions. B. cinerea releases small RNAs (sRNAs) that can suppress the expression of defense-related genes in its host plants [9]. Agrobacterium-mediated and protoplast-based transformation have been developed [12,13,14], and several vectors are available which facilitate the generation of mutants and strains expressing fluorescently tagged proteins for cytological studies [15]. The generation of multiple knock-out mutants was hampered until now by the lack of marker recycling systems for serial gene replacements, as described in some filamentous fungi [16]

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Results

Conclusion