mCherry-Labeled Verticillium dahliae Could Be Utilized to Investigate Its Pathogenicity Process in Nicotiana benthamiana

Abstract

Verticillium dahliae is a soil-borne phytopathogenic fungus that causes a destructive vascular wilt, but details of the molecular mechanism behind its pathogenicity are not very clear. Here, we generated a red fluorescent isolate of V. dahliae by protoplast transformation to explore its pathogenicity mechanism, including colonization, invasion, and extension in Nicotiana benthamiana, using confocal microscopy. The nucleotide sequences of mCherry were optimized for fungal expression and cloned into pCT-HM plasmid, which was inserted into V. dahliae protoplasts. The transformant (Vd-m) shows strong red fluorescence and its phenotype, growth rate, and pathogenicity did not differ significantly from the wild type V. dahliae (Vd-wt). Between one and three days post inoculation (dpi), the Vd-m successfully colonized and invaded epidermal cells of the roots. From four to six dpi, hyphae grew on root wounds and lateral root primordium and entered xylem vessels. From seven to nine dpi, hyphae extended along the surface of the cell wall and massively grew in the xylem vessel of roots. At ten dpi, the Vd-m was found in petioles and veins of leaves. Our results distinctly showed the pathway of V. dahliae infection and colonization in N. benthamiana, and the optimized expression can be used to deepen our understanding of the molecular mechanism of pathogenicity.

Use of red, far-red, and near-infrared light in imaging of yeasts and filamentous fungi

“…Red fluorescent protein reporters (DsRed, DsRed2, DsRed-Express, mRFP1, tdTomato, and mCherry) have also been used in a number of studies to visualize infecting hyphae of plant pathogenic fungi penetrating various plant tissues. Recent publications in this field include Penicillium digitatum (on citrus fruit, DsRed; Vu et al 2018 ), Penicillium rubens (on tomato roots, DsRed; Vallarino et al 2018 ), Verticillium dahlia (on tobacco seedlings, mCherry; Su et al 2018 ), and Epichloȅ strains (on ryegrass seedlings, DsRed; Hettiarachchige et al 2019 ). Furthermore, DsRed-labelled Verticillium longisporum was used to test the hypothesis that disease transmission may occur by seeds from European winter oilseed rape production (Zheng et al 2019 ), and the role of arthropod vectors in the propagation of grapevine trunk disease pathogenic fungi was demonstrated by the application of DsRed-Express tagged Phaeomoniella chlamydospora (Moyo et al 2014 ).…”

“…This approach may require the elaboration of new technical setups with higher intensity illumination and/or increased camera exposure time (Frigault et al 2009 ; Mubaid and Brown 2017 ). The high translucency of host tissues towards red, far-red, and infrared light in animal-fungus and plant-fungus interaction studies has also fuelled the development of new long-wavelength reporting protein systems in a number of fungal species (Leal Jr. et al 2010 ; Su et al 2018 ; Vallarino et al 2018 ). In addition, red, far-red, and infrared light have been used successfully to study host-fungus interactions deep in tissues (Bruns et al 2010 ; Hasenberg et al 2011 ; Sørensen et al 2012 ; Vasilchenko et al 2016 ; Lee et al 2018 ).…”

A droplet digital PCR assay for detection and quantification of Verticillium nonalfalfae and V. albo-atrum

“…Subsequently, hyphae infect the host through the root hairs or lateral roots ( Fradin and Thomma, 2006 ). After invading the xylem vessels, the hyphae infest the adjacent vasculature and grow along the vascular system toward the tip of the plant ( Zhao et al., 2014 ; Su et al., 2018 ). Ultimately, the pathogen reproduces and blocks the vascular system, interfering with the transport of water throughout the host and causing wilt symptoms ( Floerl et al., 2008 ; Wang et al., 2021 ).…”

Unfolded Protein Response and Scaffold Independent Pheromone MAP Kinase Signaling Control Verticillium dahliae Growth, Development, and Plant Pathogenesis

“…Unfavorable conditions induce the formation of highly resistant, black melanized microsclerotia as characteristic dormant structures which persist in the soil and can overwinter for at least 14 years [ 1 , 3 , 4 ]. The fungus germinates upon recognition of an appropriate host and enters the plant preferably via natural root wounds, root tips, or lateral root hairs [ 5 , 6 , 7 , 8 ]. Hyphae grow from cortical cells towards the central cylinder and some of them successfully reach the xylem.…”