Closed-Reference Metatranscriptomics for in Planta profiling of Fungal Pathogen Communities

Grapevines, like other perennial crops, are affected by a variety of fungal diseases that damage the trunk and other permanent structures. While in most cases individual pathogen species are considered the causal organisms of each type of trunk disease, often multiple grapevine trunk pathogens (GTPs) are recovered from infected tissues. The co-existence of different GTP species in complex and dynamic microbial communities complicates the study of the molecular mechanisms underlying disease development under vineyard conditions. The objective of this study was to develop and optimize a community-level transcriptomics (i.e., metatranscriptomics) approach that can be used to monitor simultaneously the virulence activities of multiple GTPs in planta. The availability of annotated genomes for the most common GTPs provided the unprecedented opportunity to generate a multi-species reference to be used for mapping and quantifying DNA and RNA sequencing reads for the most relevant co-infecting organisms in diseased grapevine woody tissues. We first evaluated popular sequence read mappers using permutations of multiple simulated datasets. Alignment parameters of the selected mapper were then optimized to increase the specificity and sensitivity for its application to metagenomics and metatranscriptomics analyses. Testing on grapevine woody tissues infected with individual pathogens confirmed the validity of the method. Using samples with a variety of symptoms of trunk diseases due to natural infections, we show that our approach provides quantitative assessments of species composition as well as genome-wide transcriptional profiling of potential virulence factors associated with cell wall degradation, secondary metabolism and nutrient uptake for all co-infecting pathogens.