Hex1, the Major Component of Woronin Bodies, is Required for Normal Development, Pathogenicity and Stress Response in the Plant Pathogenic FungusVerticillium 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 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.

Starvation-induced cell fusion and heterokaryosis frequently escape imperfect allorecognition systems to enable parasexual interactions in an asexual fungal pathogen

“…First, a fraction of "incompatible" interactions failed to achieve cytoplasmic mixing, which may have been prevented by a system similar to that of N. crassa, where allelic differences between individuals at the cwr locus can cause cell fusion arrest by blocking dissolution of the cell wall (Gonçalves et al, 2019). Secondly, in a fraction of "incompatible" interactions fusion triggered a cell death reaction characterized by hyphal compartmentalization, thickening of the cell wall due to increased deposition of chitin (Vangalis et al, 2020), nuclear degradation and cell shrinkage, therefore resembling the incompatibility reactions described in other fungi (Dementhon et al, 2003;Glass & Dementhon, 2006;Gonçalves et al, 2017). Such reactions are mediated by allelic interactions between sec-9/plp-1 (Heller et al, 2018) and rcd-1 (Daskalov et al, 2019) in N. crassa, as well as different repertoires of multiple het loci among fungi (Saupe, 2000;Glass & Dementhon, 2006;Paoletti, 2016).…”

“…5b,c) were obtained directly by PCR amplification from plasmid pIGPAPA, pMaM330 or pSD1 (for the sgfp, mCherry or neo R gene, respectively; Table S2) using primers containing the desired homologous arms (Table S4). Co-transformation of V. dahliae strains with plasmid pVV27 and a repair substrate was performed using a protoplast-mediated method (Vangalis et al, 2020) or the LiAc/heat shock-mediated method. Transformants were screened by PCR and the fluorescently tagged strains were confirmed by microscopy.…”

“…The two genomic amplicons and the mCherry-containing cassette were mixed in a PCR reaction without primers, which was followed by a second reaction that included the first PCR product as template and the primer pair VdH1FusF/VdH1FusR (Table S4). The fusion PCR product was then used to transform protoplasts of V. dahliae strains, according to our previously reported protocol (Vangalis et al, 2020).…”

“…a 489 bp-long fragment from plasmid pFC332 (Nødvig et al, 2015) containing the transcription termination sequence of the Aspergillus nidulans tef1 gene; and iv. a 1,648 bp-long fragment from plasmid pBS-genR (Vangalis et al, 2020) containing the G418-resistance (neo R ) cassette. The four PCR amplicons were subjected to an in vitro DNA assembly reaction in the backbone of plasmid pOSCAR (Paz et al, 2011) using the NEBuilder HiFi DNA Assembly Master Mix kit (New England Biolabs, Ipswich, MA, USA), according to the manufacturer's recommendations.…”

“…The four PCR amplicons were subjected to an in vitro DNA assembly reaction in the backbone of plasmid pOSCAR (Paz et al, 2011) using the NEBuilder HiFi DNA Assembly Master Mix kit (New England Biolabs, Ipswich, MA, USA), according to the manufacturer's recommendations. The resulting vector was used for ATMT of V. dahliae strains, according to our recently reported protocol (Vangalis et al, 2020). Nuclear labeling of V. dahliae strains with heterologous sGFP-tagged histone H1 was performed using plasmid pMF357 (Ishikawa et al, 2012).…”