In‐Plate Quantitative Characterization of Arabidopsis thaliana Susceptibility to the Fungal Vascular Pathogen Fusarium oxysporum

Abstract

Root vascular pathogens are some of the world's most devastating plant pathogens. However, the methods used to determine plant susceptibility to this class of pathogen are laborious, variable, and in most cases qualitative. Here we present a rapid, simple, and robust infection assay for the characterization of Arabidopsis thaliana resistance to the fungal root pathogen Fusarium oxysporum. The method utilizes fungal root vascular penetrations and fungal‐induced root growth inhibition to deliver a quantitative assessment of plant susceptibility with spatial and temporal resolution. These plant susceptibility indicators are paired with a semiautomated data analysis pipeline to deliver a reproducible assessment of plant susceptibility to root vascular pathogens such as F. oxysporum. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Arabidopsis thaliana plate infection assay using fluorescently labeled Fusarium oxysporum Support Protocol 1: Preparation of A. thaliana germination plates Support Protocol 2: Preparation of the F. oxysporum culture Basic Protocol 2: Data acquisition of F. oxysporum plant infection assay Support Protocol 3: Acquiring root growth inhibition data using Fiji

Impairment of the cellulose degradation machinery enhances Fusarium oxysporum virulence but limits its reproductive fitness

“…1, A and B ) ( 39 ). Therefore, we generated a CLR1 null mutant in the previously described Fo5176 pSIX1::GFP background, which allows for monitoring Fo5176 colonization of the root vasculature ( 33 , 44 ). We obtained deletion mutants lacking the entire CLR1 coding region (Δ clr1-1 and Δ clr1-2 ) and a complemented strain ( clr1C ) generated by reintroducing the wild-type (WT) pCLR1::CLR1 into the Δ clr1-1 mutant background (fig.…”

Impairment of the cellulose degradation machinery enhances fungal virulence but limits reproductive fitness

“…1A and 1B). Therefore, we generated a CLR1 null mutant in the previously described Fo5176 pSIX1::GFP background, which allows for monitoring Fo5176 colonization of the root vasculature ( 33, 41 ). We obtained deletion mutants lacking the entire CLR1 coding region ( clr1-1 and clr1-2) and a complemented strain ( clr1C) generated by reintroducing the wild-type pCLR1::CLR1 into the clr1-1 mutant background (Fig.…”

“…To determine whether Fo requires cellulose degradation to infect its plant hosts, we performed plate infection assays as described before ( 41 ). We monitored root vascular colonization by following GFP signal presence in the root vasculature at different days post-transfer (dpt) to spore-containing plates.…”

“…Arabidopsis infection assays were performed as described previously ( 33, 41 ). In summary, 8 days-old seedlings grown as described above were transplanted to plates with 100 μl of 10 7 microconidia/ml of Fo5176-pSIX1.…”

“…Unexpectedly, our data show that crystalline cellulose degradation is not only dispensable, but disadvantageous, for Fo infection, as shown by the increased virulence displayed by clr1 mutants. Using the model pathosystem Arabidopsis thaliana -Fo5176 (( 33, 34, 40, 41 ), we show that the clr1 mutant compensates for cellulose degradation deficiency by increasing the secretion of various virulence factors to compromise plant immune responses. On the other hand, the reduction of cellulose degradation capacity severely compromised fungal metabolism during its saprophytic growth, with dramatic consequences for its microconidia production.…”