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. In this study, 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. We report the development of a strategy which combines the introduction of an optimized nuclear-targeted Cas9-single guide RNA ribonucleoprotein complex (RNP) and a repair template together with unstable telomere vectors for transient selection into fungal protoplasts. A high proportion of the transformants contains the desired genetic changes, and the telomere vector is lost subsequently when selection is stopped. This system allowed introduction of changes into the genome without the requirement of selection markers. It shows superior editing efficiencies compared to existing CRISPR/Cas protocols for filamentous fungi, and leads to a very low number of additional off-target mutations. To demonstrate the performance of our protocol, we conducted for the first time a site-directed, random mutagenesis in a gene encoding an important fungicide target. This approach allows new applications such as in vivo structure-function analysis of proteins and rational fungicide resistance studies. As demonstrated with the rice blast pathogen Magnaporthe oryzae, the RNP-based CRISPR/Cas toolset with telomere vectors can be transferred to other fungi and is expected to boost their genetic manipulation.

Plant lectins are proteins that reversibly bind carbohydrates and are assumed to play an important role in plant development and resistance. Through the binding of carbohydrate ligands, lectins are involved in the perception of environmental signals and their translation into phenotypical responses. These processes require down-stream signaling cascades, often mediated by interacting proteins. Fusing the respective genes of two interacting proteins can be a way to increase the efficiency of this process. Most recently, proteins containing jacalin-related lectin (JRL) domains became a subject of plant resistance responses research. A meta-data analysis of fusion proteins containing JRL domains across different kingdoms revealed diverse partner domains ranging from kinases to toxins. Among them, proteins containing a JRL domain and a dirigent domain occur exclusively within monocotyledonous plants and show an unexpected high range of family member expansion compared to other JRL-fusion proteins. Rice, wheat, and barley plants overexpressing OsJAC1, a member of this family, are resistant against important fungal pathogens. We discuss the possibility that JRL domains also function as a decoy in fusion proteins and help to alert plants of the presence of attacking pathogens.

For plant pathogenic fungi, such as powdery mildews, that survive only on a limited number of host plant species, it is a matter of vital importance that their spores sense that they landed on the right spot to initiate germination as quickly as possible. We investigated a barley (Hordeum vulgare) mutant with reduced epicuticular leaf waxes on which spores of adapted and nonadapted powdery mildew fungi showed reduced germination. The barley gene responsible for the mutant wax phenotype was cloned in a forward genetic screen and identified to encode a 3-KETOACYL-CoA SYNTHASE (HvKCS6), a protein participating in fatty acid elongation and required for synthesis of epicuticular waxes. Gas chromatography-mass spectrometry analysis revealed that the mutant has significantly fewer aliphatic wax constituents with a chain length above C-24. Complementation of the mutant restored wild-type wax and overcame germination penalty, indicating that wax constituents less present on the mutant are a crucial clue for spore germination. Investigation of Arabidopsis (Arabidopsis thaliana) transgenic plants with sense silencing of Arabidopsis REQUIRED FOR CUTICULAR WAX PRODUCTION1, the HvKCS6 ortholog, revealed the same germination phenotype against adapted and nonadapted powdery mildew fungi. Our findings hint to an evolutionary conserved mechanism for sensing of plant surfaces among distantly related powdery mildews that is based on KCS6-derived wax components. Perception of such a signal must have been evolved before the monocot-dicot split took place approximately 150 million years ago.

Recent work has provided evidence for the occurrence of N-hydroxypipecolic acid (NHP) in Arabidopsis thaliana, characterized its pathogen-inducible biosynthesis by a three-step metabolic sequence from l-lysine, and established a central role for NHP in the regulation of systemic acquired resistance. Here, we show that NHP is biosynthesized in several other plant species in response to microbial attack, generally together with its direct metabolic precursor pipecolic acid and the phenolic immune signal salicylic acid. For example, NHP accumulates locally in inoculated leaves and systemically in distant leaves of cucumber in response to Pseudomonas syringae attack, in Pseudomonas-challenged tobacco and soybean leaves, in tomato inoculated with the oomycete Phytophthora infestans, in leaves of the monocot Brachypodium distachyon infected with bacterial (Xanthomonas translucens) and fungal (Magnaporthe oryzae) pathogens, and in M. oryzae-inoculated barley. Notably, resistance assays indicate that NHP acts as a potent inducer of acquired resistance to bacterial and fungal infection in distinct monocotyledonous and dicotyledonous species. Pronounced systemic accumulation of NHP in leaf phloem sap of locally inoculated cucumber supports a function for NHP as a phloem-mobile immune signal. Our study thus generalizes the existence and function of an NHP resistance pathway in plant systemic acquired resistance.

Plant hormones are well known regulators which balance plant responses to abiotic and biotic stresses. We investigated the role of abscisic acid (ABA) in resistance of barley (Hordeum vulgare L.) against the plant pathogenic fungus Magnaporthe oryzae. Exogenous application of ABA prior to inoculation with M. oryzae led to more disease symptoms on barley leaves. This result contrasted the finding that ABA application enhances resistance of barley against the powdery mildew fungus. Microscopic analysis identified diminished penetration resistance as cause for enhanced susceptibility. Consistently, the barley mutant Az34, impaired in ABA biosynthesis, was less susceptible to infection by M. oryzae and displayed elevated penetration resistance as compared to the isogenic wild type cultivar Steptoe. Chemical complementation of Az34 mutant plants by exogenous application of ABA re-established disease severity to the wild type level. The role of ABA in susceptibility of barley against M. oryzae was corroborated by showing that ABA application led to increased disease severity in all barley cultivars under investigation except for the most susceptible cultivar Pallas. Interestingly, endogenous ABA concentrations did not significantly change after infection of barley with M. oryzae. Our results revealed that elevated ABA levels led to a higher disease severity on barley leaves to M. oryzae. This supports earlier reports on the role of ABA in enhancing susceptibility of rice to the same pathogen and thereby demonstrates a host plant-independent function of this phytohormone in pathogenicity of monocotyledonous plants against M. oryzae.