Identifying Genes in Fusarium verticillioides Through Forward and Reverse Genetics

Abstract

The increasing availability of sequenced genomes for plant pathogenic fungi has revolutionized molecular plant pathology in recent years. However, the genetic regulatory networks underlying many important components of pathogenesis remain poorly defined. Although the protocols outlined in this chapter can be utilized to identify genes regulating a wide range of biological processes in many filamentous fungi, we focus on describing how to identify genes through forward and reverse genetics, using the plant pathogenic fungus Fusarium verticillioides as a model for the protocol. Specifically, this chapter explains how to create a collection of insertional mutants via Restriction Enzyme Mediated Integration (REMI) and how to screen mutants with a high-throughput method to visualize defects in amylolysis. Next, techniques are described to define the genomic lesions in REMI mutants with genome-walker PCR in order to identify candidate genes. Finally, protocols are presented describing a reverse-genetic approach to disrupt candidate genes in the wild-type strain with a split-marker strategy to confirm the phenotype observed in the REMI mutant.

Involvement of FST1 from Fusarium verticillioides in virulence and transport of inositol

“…The resulting split‐marker products were concentrated by ethanol precipitation and used to transform protoplasts of FvWT, as described by Ridenour et al . (). Hygromycin‐resistant transformants were screened by PCR with primers FST1_A1/HYGSCRN_B.…”

“…Generation of the Dfst1 strain FST1 in F. verticillioides was deleted by the split-marker method described by Ridenour et al (2012). Upstream and downstream fragments of FST1 were amplified by polymerase chain reaction (PCR) with the primer pairs FST1_F1/FST1_F2 and FST1_F3/FST1_F4 (Table S2, see Supporting Information), respectively.…”

“…This was followed by 948C for 1 min, 35 cycles of 948C for 30 s, 608C for 30 s and 728C for 140 s, and one cycle of 728C for 5 min. The resulting splitmarker products were concentrated by ethanol precipitation and used to transform protoplasts of FvWT, as described by Ridenour et al (2012). Hygromycin-resistant transformants were screened by PCR with primers FST1_A1/HYGSCRN_B.…”

UBL1 of Fusarium verticillioides links the N‐end rule pathway to extracellular sensing and plant pathogenesis

“…Protoplasts for F. graminearium , F. verticillioides and F. oxysporum f. sp. lycopersici were obtained and transformed following standard protocols (Hou et al ., ; Ridenour et al ., ).…”

“…Thus, UBL1 is not required for the transition from random to directed growth during silk colonization, but disruption of UBL1 significantly impaired the rate of directional growth. Virulence on wheat was initially evaluated by spraying wheat heads with spore suspensions of F. graminearum strains PH-1, UFG-1, UFG-2 or UFG-4 to mimic natural infection conditions and provide uniform inoculations (Horevaj and Bluhm, 2012). Disease development was evaluated daily from 4 to 7 days after inoculation ( Fig.…”