CRISPR-Cas9 gene editing and rapid detection of gene-edited mutants using high-resolution melting in the apple scab fungus, Venturia inaequalis.

Apple scab, caused by the fungal pathogen Venturia inaequalis, is the most economically important disease of apple (Malus x domestica) worldwide. To develop durable control strategies against this disease, a better understanding of the genetic mechanisms underlying the growth, reproduction, virulence and pathogenicity of V. inaequalis is required. A major bottleneck for the genetic characterization of V. inaequalis is the inability to easily delete or disrupt genes of interest using homologous recombination. Indeed, no gene deletions or disruptions in V. inaequalis have yet been published. Using the melanin biosynthesis pathway gene trihydroxynaphthalene reductase (THN) as a target for inactivation, which has previously been shown to result in a light-brown colony phenotype when transcriptionally silenced using RNA interference, we show, for the first time, that the CRISPR-Cas9 gene editing system can be successfully applied to the apple scab fungus. More specifically, using a CRISPR-Cas9 single guide RNA (sgRNA) targeted to the THN gene, delivered by a single autonomously replicating Golden Gate-compatible plasmid, we were able to identify six of 36 stable transformants with a light-brown phenotype, indicating an ∼16.7% gene inactivation efficiency. Notably, of the six THN mutants, five had an independent mutation. As part of our pipeline, we also report a high-resolution melting (HRM) curve protocol for the rapid detection of CRISPR-Cas9 gene-edited mutants of V. inaequalis. This protocol identified a single base pair deletion mutation in a sample containing only 5% mutant genomic DNA, indicating high sensitivity for mutant screening. In establishing CRISPR-Cas9 as a tool for gene editing in V. inaequalis, we have provided a strong starting point for studies aiming to decipher gene function in this fungus. The associated HRM curve protocol will enable CRISPR-Cas9 transformants to be screened for gene inactivation in a high-throughput and low-cost manner, which will be particularly powerful in cases where the CRISPR-Cas9-mediated gene inactivation efficiency is low.

Dissection of the epoxyjanthitrem pathway in Epichloë sp. LpTG-3 strain AR37 by CRISPR gene editing.

Epichloë festucae var. lolii and Epichloë sp. LpTG-3 are filamentous fungal endophytes of perennial ryegrass (Lolium perenne) that have a substantial impact on New Zealand's agricultural economy by conferring biotic advantages to the host grass. Overall, Epichloë endophytes contribute NZ$200 million to the economy annually, with strain AR37 estimated to contribute NZ$3.6 billion to the New Zealand economy over a 20-year period. This strain produces secondary metabolites, including epoxyjanthitrems, which are a class of indole diterpenes, associated with the observed effects of AR37 on livestock and insect pests. Until very recently, AR37 was intractable to genetic modification but this has changed with the application of CRISPR-Cas9 based gene editing techniques. In this paper, gene inactivation by CRISPR-Cas9 was used to deconvolute the genetic basis for epoxyjanthitrem biosynthesis, including creating an AR37 strain that has been edited to remove the biosynthesis of all indole diterpenes. We show that gene editing of Epichloë can be achieved without off-target events or introduction of foreign DNA (footprint-less) through an AMA1-based plasmid that simultaneously expresses the CRISPR-Cas9 system and selectable marker. Genetic modification events in these transformants were investigated through genome sequencing and in planta chemistry.

Genetic Manipulation of the Ergot Alkaloid Pathway in Epichloë festucae var. lolii and Its Effect on Black Beetle Feeding Deterrence.

Epichloë endophytes are filamentous fungi (family Clavicipitaceae) that live in symbiotic associations with grasses in the sub family Poöideae. In New Zealand, E. festucae var. lolii confers significant resistance to perennial ryegrass (Lolium perenne) against insect and animal herbivory and is an essential component of pastoral agriculture, where ryegrass is a major forage species. The fungus produces in planta a range of bioactive secondary metabolites, including ergovaline, which has demonstrated bioactivity against the important pasture pest black beetle, but can also cause mammalian toxicosis. We genetically modified E. festucae var. lolii strain AR5 to eliminate key enzymatic steps in the ergovaline pathway to determine if intermediate ergot alkaloid compounds can still provide insecticidal benefits in the absence of the toxic end product ergovaline. Four genes (dmaW, easG, cloA, and lpsB) spanning the pathway were deleted and each deletion mutant was inoculated into five different plant genotypes of perennial ryegrass, which were later harvested for a full chemical analysis of the ergot alkaloid compounds produced. These associations were also used in a black beetle feeding deterrence study. Deterrence was seen with just chanoclavine present, but was cumulative as more intermediate compounds in the pathway were made available. Ergovaline was not detected in any of the deletion associations, indicating that bioactivity towards black beetle can be obtained in the absence of this mammalian toxin.

Seed Transmission of Epichloë Endophytes in Lolium perenne Is Heavily Influenced by Host Genetics.

Vertical transmission of symbiotic Epichloë endophytes from host grasses into progeny seed is the primary mechanism by which the next generation of plants is colonized. This process is often imperfect, resulting in endophyte-free seedlings which may have poor ecological fitness if the endophyte confers protective benefits to its host. In this study, we investigated the influence of host genetics and environment on the vertical transmission of Epichloë festucae var. lolii strain AR37 in the temperate forage grass Lolium perenne. The efficiency of AR37 transmission into the seed of over 500 plant genotypes from five genetically diverse breeding populations was determined. In Populations I-III, which had undergone previous selection for high seed infection by AR37, mean transmission was 88, 93, and 92%, respectively. However, in Populations IV and V, which had not undergone previous selection, mean transmission was 69 and 70%, respectively. The transmission values, together with single-nucleotide polymorphism data obtained using genotyping-by-sequencing for each host, was used to develop a genomic prediction model for AR37 seed transmission. The predictive ability of the model was estimated at r = 0.54. While host genotype contributed greatly to differences in AR37 seed transmission, undefined environmental variables also contributed significantly to seed transmission across different years and geographic locations. There was evidence for a small host genotype-by-environment effect; however this was less pronounced than genotype or environment alone. Analysis of endophyte infection levels in parent plants within Populations I and IV revealed a loss of endophyte infection over time in Population IV only. This population also had lower average tiller infection frequencies than Population I, suggesting that AR37 failed to colonize all the daughter tillers and therefore seeds. However, we also observed that infection of seed by AR37 may fail during or after initiation of floral development from plants where all tillers remained endophyte-infected over time. While the effects of environment and host genotype on fungal endophyte transmission have been evaluated previously, this is the first study that quantifies the relative impacts of host genetics and environment on endophyte vertical transmission.

Identification of the dialysable serum inducer of germ-tube formation in Candida albicans.

Yeast cells of Candida albicans are induced by serum at 37 degrees C to produce germ tubes, the first step in a transition from yeast to hyphal growth. Previously, it has been shown that the active component is not serum albumin but is present in the dialysable fraction of serum. In this study, serum induction of germ-tube formation is shown to occur even in the presence of added exogenous nitrogen sources and is therefore not signalled by nitrogen derepression. The active component in serum was purified by ion-exchange, reverse-phase and size-exclusion chromatography from the dialysable fraction of serum and was identified by NMR to be d-glucose. Enzymic destruction of glucose, using glucose oxidase, demonstrated that d-glucose was the only active component in these fractions. Induction of germ-tube formation by d-glucose required a temperature of 37 degrees C and the pH optimum was between pH 7.0 and 8.0. d-Glucose induced germ-tube formation in a panel of clinical isolates of C. albicans. Although d-glucose is the major inducer in serum, a second non-dialysable, trichloroacetic acid precipitable inducer is also present. However, whereas either 1.4 % (v/v) serum or an equivalent concentration of d-glucose induced 50 % germ-tube formation, the non-dialysable component required a 10-fold higher concentration to induce 50 % germ-tube formation. Serum is, therefore, the most effective induction medium for germ-tube formation because it is buffered at about pH 8.5 and contains two distinct inducers (glucose and a non-dialysable component), both active at this pH.