Johnson, L. J. | bioRxiv Science

bioRxiv2021

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

BackgroundScab, or black spot, caused by the filamentous 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. Recently, CRISPR-Cas9 has emerged as an efficient tool for gene editing in filamentous fungi. With this in mind, we set out to establish CRISPR-Cas9 as a gene editing tool in V. inaequalis. ResultsWe showed that CRISPR-Cas9 can be used for gene inactivation in the apple scab fungus. As a proof of concept, we targeted the melanin biosynthesis pathway gene trihydroxynaphthalene reductase (THN), which has previously been shown to result in a light-brown colony phenotype when transcriptionally silenced using RNA interference. Using one of two CRISPR-Cas9 single guide RNAs (sgRNAs) 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 these 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. ConclusionsIn establishing CRISPR-Cas9 as a tool for gene editing in V. inaequalis, we have provided a strong starting point for studies aiming to decipher the function of genes associated with the growth, reproduction, virulence and pathogenicity of 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.

microbiology↗

bioRxiv2019

Homologues of key circadian clock genes present in Verticillium dahliae do not direct circadian programs of development or mRNA abundance.

Many organisms harbour circadian clocks that promote their adaptation to the rhythmic environment. While a broad knowledge of the molecular mechanism of circadian clocks has been gained through the fungal model Neurospora crassa, little is known about circadian clocks in other fungi. N. crassa belongs to the same class as many important plant pathogens including the vascular wilt fungus Verticillium dahliae. We identified homologues of N. crassa clock proteins in V. dahliae, which showed high conservation in key protein domains. However, no evidence for an endogenous, free-running and entrainable rhythm was observed in the daily formation of conidia and microsclerotia. In N. crassa the frequency (frq) gene encodes a central clock protein expressed rhythmically and in response to light. In contrast, expression of Vdfrq is not light-regulated. Temporal gene expression profiling over 48 hours in constant darkness and temperature revealed no circadian expression of key clock genes. Furthermore, RNA-seq over a 24 h time-course revealed no robust oscillations of RNA in constant darkness. Comparison of gene expression between wild-type V. dahliae and a{Delta} Vdfrq mutant showed that genes involved in metabolism, transport and redox processes are mis-regulated in the absence of Vdfrq. In addition, Vd{Delta}frq mutants display growth defects and reduced pathogenicity in a strain dependent manner. Our data indicate that if a circadian clock exists in Verticillium, it is based on alternative mechanisms such as post-transcriptional interactions of VdFRQ and the WC proteins or the components of a FRQ-less oscillator. Alternatively, it could be that whilst the original functions of the clock proteins have been maintained, in this species the interactions that generate rhythmicity have been lost or are only triggered when specific environmental conditions are met. The presence of conserved clock genes in genomes should not be taken as definitive evidence of circadian function. Author summaryCircadian clocks are used by organisms to orchestrate the activity of cellular processes such that they occur at an optimal time of day. Research carried out in the filamentous fungus Neurospora crassa has revealed a huge amount of information about the components its circadian clock, its interactions with the environment and how it drives cellular biochemistry and physiology. Although homologues of the Neurospora clock genes are present in a number of fungi, functional clocks have been demonstrated in a just a handful. Importantly, a link between the circadian clock of the plant pathogen Botrytis cinerea and virulence has recently been reported. We report that another significant plant pathogen, Verticillium dahliae, contains well-conserved homologues of all key clock genes. We find that diurnal development of conidia and microsclerotia is not influenced by a circadian clock. Furthermore, in a constant environment we find no evidence of rhythmic transcript accumulation. However, deletion of the central clock component results in altered growth and reduced virulence. This led us to question the role of clock genes in Verticillium. We are forced to consider that in this species the interactions that generate rhythmicity have been lost, are generated purely via post-transcriptional modification of clock proteins, are only triggered when specific environmental conditions are met or never evolved.

microbiology↗