In Vitro Secretome Analysis Suggests Differential Pathogenic Mechanisms between Fusarium oxysporum f. sp. cubense Race 1 and Race 4

Abstract

Banana Fusarium wilt, caused by the fungus pathogen Fusarium oxysporum f. sp. cubense (Foc), is a devastating disease that causes tremendous reductions in banana yield worldwide. Secreted proteins can act as pathogenicity factors and play important roles in the Foc–banana interactions. In this study, a shotgun-based proteomic approach was employed to characterize and compare the secretomes of Foc1 and Foc4 upon banana extract treatment, which detected 1183 Foc1 and 2450 Foc4 proteins. Comprehensive in silico analyses further identified 447 Foc1 and 433 Foc4 proteins in the classical and non-classical secretion pathways, while the remaining proteins might be secreted through currently unknown mechanisms. Further analyses showed that the secretomes of Foc1 and Foc4 are similar in their overall functional characteristics and share largely conserved repertoires of CAZymes and effectors. However, we also identified a number of potentially important pathogenicity factors that are differentially present in Foc1 and Foc4, which may contribute to their different pathogenicity against banana hosts. Furthermore, our quantitative PCR analysis revealed that genes encoding secreted pathogenicity factors differ significantly between Foc1 and Foc4 in their expression regulation in response to banana extract treatment. To our knowledge, this is the first experimental secretome analysis that focused on the pathogenicity mechanism in different Foc races. The results of this study provide useful resources for further exploration of the complicated pathogenicity mechanisms in Foc.

FoCupin1, a Cupin_1 domain-containing protein, is necessary for the virulence of Fusarium oxysporum f. sp. cubense tropical race 4

“…Plant pathogens employ effectors as molecular weapons to manipulate host immunity and facilitate colonization ( Bi and Zhou, 2021 ). Recent studies have shown that the effector proteins secreted by Foc4 can act as pathogenicity factors and play important roles in Foc-banana interaction ( Li et al, 2019 ; He et al, 2021 ). Almost 1,000 putative secreted proteins have been predicted in Foc genome, many of which are thought to be candidates for effector proteins ( Guo et al, 2015 ).…”

“…The wild-type strain and the transformants generated in this study were grown at 28°C on PDA medium for 5 days to assess growth and colony characteristics. Czapek Dox (CD) medium was used for conidiation assays and liquid NCMB medium was used to mimic the Foc-banana interaction as described ( He et al, 2021 ). The Foc TR4-susceptible banana cultivar Brazilian (AAA group, Cavendish) and tobacco ( Nicotiana benthamiana ) were used in this study.…”

“…Five internet-based tools, SignalP 4.0, WoLF PSORT, TargetP 1.1, TMHMM Server 2.0, and big-PI predictor, were employed to analyze FoCupin1 as described ( He et al, 2021 ). The candidate effector analysis was predicted using the online predictor tool EffectorP 2.0 1 with setting the class probability threshold to 0.5 ( Sperschneider et al, 2018 ).…”

“…Total RNA was extracted from Foc TR4 using a Fungal RNA kit (Omega, United States) according to the manufacturer’s protocol as described previously ( He et al, 2021 ). Briefly, banana roots were grounded thoroughly with liquid nitrogen.…”

“…In our previous work, a shotgun-based proteomic approach was employed to identify the secreted proteins of Foc TR4, followed by high-quality secretome prediction and in-depth in silico analysis, which provided a resource for studying Foc TR4 candidate effectors ( He et al, 2021 ). Here, we analyzed a candidate effector, a Cupin type-1 domain-containing protein (named as FoCupin1), using the Agrobacterium -mediated transient expression system in tobacco.…”