Mining the penicillium expansum genome for virulence genes: A functional-based approach to discover novel loci mediating blue mold decay of apple fruit

Authors: LUCIANO-ROSARIO, DIANIRIS - Oak Ridge Institute For Science And Education (ORISE); PENG, HUI - University Of Florida; Gaskins, Verneta; Fonseca, Jorge; KELLER, NANCY - University Of Wisconsin; Jurick, Wayne

Submitted to: The Journal of Fungi
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 10/28/2023
Publication Date: 11/1/2023
Citation: Luciano-Rosario, D., Peng, H., Gaskins, V.L., Fonseca, J.M., Keller, N.P., Jurick II, W.M. 2023. Mining the penicillium expansum genome for virulence genes: A functional-based approach to discover novel loci mediating blue mold decay of apple fruit. The Journal of Fungi. 9(11). Article e1066. https://doi.org/10.3390/jof9111066.
DOI: https://doi.org/10.3390/jof9111066

Interpretive Summary: Blue mold is a postharvest disease of apples and pears that causes significant economic losses and threatens food safety and security. This disease is mainly caused by a filamentous fungus that also thrives in apple storage conditions. There are limited methods to control blue mold, the main one being the use of fungicides. To develop new control methods, there is a need to understand and identify potential genes that allow the fungus to cause apple fruit decay. Hence, we generated mutants that had different genes interrupted to test if these were important for blue mold decay when they were inoculated into apple fruit. We identified 5 new important genes and analyzed their involvement in blue mold decay. Ultimately, this study expands the potential to develop novel control strategies for blue mold using biotechnology approaches and serves as a platform for others in the scientific community to examine an unlimited range of biological questions using this mutant collection.

Technical Abstract: Blue mold, an economically impactful postharvest disease of pome fruits, is caused by the mycotoxigenic fungus Penicillium expansum. In addition to the economic losses caused by P. expansum, food safety can be compromised as this pathogen produces patulin, a mycotoxin regulated by many government agencies worldwide. Although there have been many advances in understanding the molecular biology of P. expansum, there is a need to unravel this pathogen’s disease strategies to develop additional decay and toxin management tools. In this study, forward and reverse genetic approaches were used to identify genes involved in blue mold infection biology in apple fruits. For this, the highly aggressive P. expansum R19 strain was transformed with Agrobacterium tumefaciens to generate a random T-DNA insertional mutant library. A total of 448 transformants were generated, single-spore propagated, and screened for reduced decay phenotype on inoculated apple fruits. The mutants ranged in disease severity from wild type (WT) levels up to a 20% reduction in lesion diameter. Of these mutants, six (T-193, T-275, T-434, T-588, T-625, and T-711) were selected and five unique genes were identified of interest via TAIL-PCR. Southern Blot hybridization revealed single insertional events where the T-DNA integrated into non-coding regions except in T-625. Gene expression levels of the WT strain confirmed that the identified genes were transcribed both in vitro and in vivo. To further characterize these genes and their role in blue mold decay, two deletion mutants ('t625 and 't588) and a knock-down strain (t-434KD) were generated for three loci. Two mutants (T-193, T-275) loci were recalcitrant to either deletion or knock-down approaches. Data shows that the 't588 mutant phenocopied the T-DNA insertion strains and has virulence penalties during apple fruit decay. We hypothesize that this locus encodes a glyoxalase due to the predicted function from bioinformatic analyses and the reduced diameter of colonies grown in methyl glyoxylate (MG). This work presents novel members of signaling networks and additional genetic factors that regulate fungal virulence in the blue mold fungus during apple fruit decay.