A single laccase acts as a key component of environmental sensing in a broad host range fungal pathogen.

Secreted laccases are important enzymes on a broad ecological scale for their role in mediating plant-microbe interactions, but within ascomycete fungi these enzymes have been primarily associated with melanin biosynthesis. In this study, a putatively secreted laccase, Sslac2, was characterized from the broad-host-range plant pathogen Sclerotinia sclerotiorum, which is largely unpigmented and is not dependent on melanogenesis for plant infection. Gene knockouts of Sslac2 demonstrate wide ranging developmental phenotypes and are functionally non-pathogenic. These mutants also displayed indiscriminate growth behaviors and enhanced biomass formation, seemingly as a result of their inability to respond to canonical environmental growth cues, a phenomenon further confirmed through chemical stress, physiological, and transcriptomic analyses. Transmission and scanning electron microscopy demonstrate apparent differences in extracellular matrix structure between WT and mutant strains that likely explain the inability of the mutants to respond to their environment. Targeting Sslac2 using host-induced gene silencing significantly improved resistance to S. sclerotiorum, suggesting that fungal laccases could be a valuable target of disease control. Collectively, we identified a laccase critical to the development and virulence of the broad-host-range pathogen S. sclerotiorum and propose a potentially novel role for fungal laccases in modulating environmental sensing.

Analytic and Clinical Validation of a Pan-Cancer NGS Liquid Biopsy Test for the Detection of Copy Number Amplifications, Fusions and Exon Skipping Variants.

Liquid biopsies are an integral part of the diagnosis of cancer. Here, we have extended previous validation studies of a new targeted NGS panel to include the detection of copy number amplifications (CNAs), fusions, and exon skipping variants. Detection of these gene classes included specimens from clinical and healthy donors and cell lines (fusions: ROS1, EML4-ALK, NTRK1; exon skipping: MET exon 14; CNAs: HER2, CDK6, EGFR, MYC, and MET). The limit of detection (LOD) for fusion/skipping was 42 copies (QC threshold was three copies) and was verified using three additional fusion/skipping variants. LOD for CNAs was 1.40-fold-change (QC threshold = 1.15-fold change) and was verified with three additional CNAs. In repeatability and intermediate precision (within lab) studies, all fusion/skipping variants were detected in all runs and all days of testing (n = 18/18; 100%); average CV for repeatability was 20.5% (range 8.7-34.8%), and for intermediate precision it was 20.8% (range 15.7-30.5%). For CNAs, 28/29 (96.6%) copy gains were detected. For CNAs, the average CV was 1.85% (range 0% to 5.49%) for repeatability and 6.59% (range 1.65% to 9.22%) for intermediate precision. The test panel meets the criteria for being highly sensitive and specific and extends its utility for the serial detection of clinically relevant variants in cancer.