Hex1, the Major Component of Woronin Bodies, Is Required for Normal Development, Pathogenicity, and Stress Response in the Plant Pathogenic Fungus Verticillium dahliae

Abstract

Woronin bodies are membrane-bound organelles of filamentous ascomycetes that mediate hyphal compartmentalization by plugging septal pores upon hyphal damage. Their major component is the peroxisomal protein Hex1, which has also been implicated in additional cellular processes in fungi. Here, we analyzed the Hex1 homolog of Verticillium dahliae, an important asexual plant pathogen, and we report its pleiotropic involvement in fungal growth, physiology, stress response, and pathogenicity. Alternative splicing of the Vdhex1 gene can lead to the production of two Hex1 isoforms, which are structurally similar to their Neurospora crassa homolog. We show that VdHex1 is targeted to the septum, consistently with its demonstrated function in sealing hyphal compartments to prevent excessive cytoplasmic bleeding upon injury. Furthermore, our investigation provides direct evidence for significant contributions of Hex1 in growth and morphogenesis, as well as in asexual reproduction capacity. We discovered that Hex1 is required both for normal responses to osmotic stress and factors that affect the cell wall and plasma-membrane integrity, and for normal resistance to oxidative stress and reactive oxygen species (ROS) homeostasis. The Vdhex1 mutant exhibited diminished ability to colonize and cause disease on eggplant. Overall, we show that Hex1 has fundamentally important multifaceted roles in the biology of V. dahliae.

Establishment of conidial fusion in the asexual fungus Verticillium dahliae as a useful system for the study of non-sexual genetic interactions

“…Translocation of nuclei through septae in CATs was never observed, indicating that those CATs that develop a septum experience a translocation-competent phase of limited duration, which is completed with the septum formation. These findings are in agreement with our previous observations of nuclear migration through septal pores being extremely rare in V. dahliae (Vangalis et al 2020).…”

“…These are triggered by genetic differences of the two nuclei and would typically result either in the prevention of fusion or in the destruction of the heterokaryotic cell (Gonçalves and Glass 2020). It is possible that in fungi such as V. dahliae, which almost exclusively have one nucleus per cellular compartment and nuclei cannot normally migrate through septal pores (Vangalis et al 2020), the observed degradation of the additional nucleus in the dikaryon might be the result of a homeostatic mechanism, possibly involving autophagy (Corral-Ramos et al 2015), that strictly regulates the number of nuclei per cell. In any case, however, one can speculate that even if nuclear degradation is a general phenomenon, the co-existence of nuclei for up to several hours before the onset of degradation, and the presence of chromatin of the degraded nucleus in the cell after nuclear disorganization provide possible windows of opportunity for genetic interactions of the two nuclei, as has been previously discussed (He et al 1998;Richards et al 2011;Vlaardingerbroek et al 2016).…”

“…All plasmids constructed and used for fungal transformation are listed in Table S3. The hypervirulent Agrobacterium tumefaciens strain AGL-1 was transformed with appropriate binary vectors according to previously reported procedures (Vangalis et al 2020). We followed our optimized and published protocols for protoplast and A. tumefaciens-mediated transformation of V. dahliae (Vangalis et al 2020).…”

“…For tagging of histone H1 with sGFP, we transformed protoplasts of V. dahliae isolates Ls.17 and Cf.38 with plasmid pMF357 (Table S3), which contains the hph cassette that confers resistance to hygromycin B, as well as a fusion construct of the sGFP gene to the N. crassa histone H1 gene. The V. dahliae Ls.17 strain with robust cytoplasmic expression of sGFP has been described before (Vangalis et al 2020).…”

The NADPH Oxidase A of Verticillium dahliae is Essential for Pathogenicity, Normal Development, and Stress Tolerance, and it Interacts with Yap1 to Regulate Redox Homeostasis

“…We have recently described the strategy that we used for knocking out the V. dahliae homologs of noxA and yap1 [23]. Briefly, both mutants were constructed in the background of V. dahliae wild-type strain 123V via double homologous recombination following Agrobacterium tumefaciens -mediated transformation (ATMT) [47]. Using this method, we recently performed and described deletion of noxA and validation of the mutant strain [23].…”