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 RNPbased cotransformation is expected to accelerate molecular research in B. cinerea and other fungi.

Summary Magnaporthe oryzae secretes several effectors that modulate and hijack rice processes to colonize host cells, but the underlying mechanisms remain unclear. We report on a novel cytoplasmic effector MoIug4 that targets the rice ethylene pathway as a transcription repressor to subvert host immunity. We found that MoIug4 binds to the promoter of the host OsEIN2 gene that encodes a central signal transducer in the ethylene‐signaling pathway. We also identified a MoIug4 interacting protein, OsAHL1, which acts as an AT‐hook motif‐containing protein binding to the A/T‐rich promoter regions. Our knockout and overexpression studies showed that OsAHL1 positively regulates plant immunity in response to M. oryzae infection. OsAHL1 exhibits transcriptional regulatory activities by binding the OsEIN2 promoter region, similar to MoIug4. Intriguingly, we found that MoIug4 exhibits a higher binding affinity than OsAHL1 to the OsEIN2 promoter, suggesting differential regulatory specificities. These results revealed a counter‐defense strategy by which the pathogen effector suppresses the activation of host defense genes by interfering with host transcription activator functions.

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Wheat blast caused by Magnaporthe oryzae pathotype Triticum (MoT), initially restricted to South America, is a global threat for wheat after spreading to Asia in 2016 by the introduction of contaminated seeds, raising the question about transmission of the pathogen from seeds to seedlings, a process so far not well understood. We therefore studied the relationship between seed infection and disease symptoms on seedlings and adult plants. To accomplish this objective, we inoculated spikes of wheat cv. Apogee with a transgenic isolate (MoT‐DsRed, with the addition of being resistant to hygromycin). We identified MoT‐DsRed in experiments using hygromycin resistance for selection or by observation of DsRed fluorescence. The seeds from infected plants looked either apparently healthy or shrivelled. To evaluate the transmission, two experimental designs were chosen (blotter test and greenhouse) and MoT‐DsRed was recovered from both. This revealed that MoT is able to colonize wheat seedlings from infected seeds under the ground. The favourable conditions of temperature and humidity allowed a high recovery rate of MoT from wheat shoots when grown in artificial media. Around 42 days after germination of infected seeds, MoT‐DsRed could not be reisolated, indicating that fungal progression, at this time point, did not proceed systemically/endophytically. We hypothesize that spike infection might occur via spore dispersal from infected leaves rather than within the plant. Because MoT‐DsRed was not only successfully reisolated from seed coats and germinating seeds with symptoms, but also from apparently healthy seeds, urgent attention is needed to minimize the risks of inadvertent dispersal of inoculum.

20CRISPR/Cas has become the state-of-the-art technology for genetic manipulation in diverse 21 organisms, enabling targeted genetic changes to be performed with unprecedented 22 37 research in B. cinerea and other fungi. 38 39 40Botrytis cinerea is a plant pathogenic ascomycete which infects more than a thousand species, 41 triggering gray mold disease which is responsible for over a billion dollars of losses in fruits, vegetables 42 and flowers every year [1]. Due to its worldwide occurrence, great economic importance and non-43 specific necrotrophic lifestyle, it has been ranked as the second most important plant pathogenic 44 fungus [2]. Control of gray mold often requires repeated treatments with fungicides, in particular 45 under high humidity conditions, but rapid adaption and resistance development of B. cinerea has 46 dramatically reduced their efficiency worldwide in many cultures, for example in strawberry fields [3]. 47After germination of a conidium on the plant surface, the fungus penetrates and invades the host, 48 rapidly killing plant cells by releasing a complex mixture of cell wall degrading enzymes, phytotoxic 49 metabolites and proteins, and by tissue acidification [4, 5]. How host cell death is induced is not fully 50 understood, but the invading hyphae seem to trigger the hypersensitive response, a plant-specific type 51 of apoptosis linked to strong defence reactions [6, 7]. Furthermore, B. cinerea releases small RNAs 52 (sRNAs) that can suppress the expression of defence-related genes in its host plants [8]. As a 53 countermeasure, plants also release sRNAs aimed to suppress fungal virulence [9]. To facilitate access 54 to genes or non-coding RNA loci that are important for pathogenesis, a gapless genome sequence of 55 B. cinerea has been published recently [10]. Considerable efforts have been made to generate tools 56 for the genetic manipulation of B. cinerea. Agrobacterium-mediated and protoplast-based 57 transformation have been developed [11][12][13], and several vectors are available which facilitate the 58 generation of mutants and strains expressing fluorescently tagged proteins for cytological studies [14]. 59Nevertheless, the generation of mutants remains time-consuming, partly because of the multinuclear 60 nature of B. cinerea, which requires several rounds of sub-cultivation to achieve homokaryosis. 61 Furthermore, the generation of multiple knock-out mutants is hampered by the lack of marker 62 recycling systems for serial gene replacements, as described in some filamentous fungi [15]. 63The application of the clustered regularly interspaced short palindromic repeats (CRISPR)-associated 64 RNA-guided Cas9 endonuclease activity has revolutionized genome editing and greatly facilitated the 65 4 genetic manipulation in a wide range of species [16]. CRISPR/Cas is based on the introduction of double 66 stranded breaks by the Cas9 endonuclease in the genome of an organism. Cas9 targeting occurs by 67 complementary sequences of a single guide RNA (sgRNA), which directs the endonu...