BZcon1, a SANT/Myb-type gene involved in the conidiation of Cochliobolus carbonum

Abstract

The fungal pathogen Cochliobolus carbonum (anamorph, Bipolaris zeicola) causes Northern Leaf Spot, leading to a ubiquitous and devastating foliar disease of corn in Yunnan Province, China. Asexual spores (conidia) play a major role in both epidemics and pathogenesis of Northern Leaf Spot, but the molecular mechanism of conidiation in C. carbonum has remained elusive. Here, using a map-based cloning strategy, we cloned a single dominant gene, designated as BZcon1 (for Bipolaris zeicola conidiation), which encodes a predicted unknown protein containing 402 amino acids, with two common conserved SANT/Myb domains in N-terminal. The BZcon1 knockout mutant completely lost the capability to produce conidiophores and conidia but displayed no effect on hyphal growth and sexual reproduction. The introduced BZcon1 gene fully complemented the BZcon1 null mutation, restoring the capability for sporulation. These data suggested that the BZcon1 gene is essential for the conidiation of C. carbonum.

Keywords: Bipolaris zeicola; Northern Leaf Spot; clone; conidia; sporulation.

Figure 1

Genetic and physical maps around the BZcon1 locus in the isolate Aa113. Genetic maps were constructed using multipoint linkage analysis on segregating populations of 271 progeny with the Mapmaker/Exp 3.0 software. Dashed lines connect the physical location of markers to their location on the genetic map or connect markers to their location in the bacterial artificial chromosome (BAC) clone contig. The gene was predicted according to Fgenesh programs and the arrows correspond to position of gene candidates. CAN13 is the gene identified as a candidate for BZcon1 and used for knockout and complementation assays.

Figure 2

Strategy for BZcon1 gene knockout and confirmation of BZcon1 mutant. (A) BZcon1 knockout vector pBSKC1 contains a 2.0-kb hygB resistance gene cassette. Homologous recombination through a double crossover event results in the knockout of a BZcon1 region with the hygB resistance cassette (hph). This region includes a 634-bp-long upstream fragment, the entire open reading frame (1209-bp-long) of BZcon1 gene, and a 76-bp-long downstream fragment. The positions of primers are indicated by small arrows. X, XhoI. H, HindIII. E, EcoRI. (B) Southern blot analysis. Total genomic DNA samples isolated from Aa113 (experimental isolate) and M113-1 (BZcon1 knockout mutant of Aa113) were digested with SalI. The hph prober, a 1-kb PCR fragment amplified from pBSKC1 using primers H-F2 and H-R2, is exactly the BZcon1 region replaced by the hygB resistance gene. (C) Total RNA extracted from the mycelia of Aa113, M113-1, and C113-2 grown for 3 to 8 d on PDA plates, respectively. And obtained RNA was subjected to RT-PCR using BZcon1 gene-specific primers CF1 and CR1. The RT-PCR product is an approximately 1.2-kb fragment in Aa113 and C113-1 (BZcon1 complemented transformant of M113-1) as predicted, but is missing in M113-1.

Figure 3

Mating result of the BZcon1 knocked-out mutant on Sachs agar medium. Pseudothecia is visible as dark spots. S92, field isolate, MAT1-2-1; S129, field isolate, MAT1-1-1; Aa113, experimental isolate, MAT1-2-1; Aa82, experimental isolate, MAT1-2-1; Aa108, experimental isolate, MAT1-1-1; and M113-1, MAT1-2-1, BZcon1 knockout mutant of Aa113.

Figure 4

Phenotypes of the wild-type strain Aa113 and S129, BZcon1 knockout mutant M113-1, BZcon1 complemented transformants C113-2 and C129-1. (A) Colony morphology of Aa113, S129, M113-1, C113-2, and C129-1 on potato dextrose agar (PDA) plates inoculated for 6 d. (B) Plate cultures observed under light microscope (red arrows showing conidia).

Figure 5

Sequence comparisons of the upstream region of BZcon1 from two isolates A113 and S129. Dot line with double arrows shows the absence of 126 continuous nucleotides in the upstream (+426 bp) of BZcon1 from the isolate S129 when compared with that of Aa113.

Figure 6

An alignment of the amino acid sequences of BZCON1 and its 11 related homologs. Two SANT/Myb-like domains were located at N-terminal (approximately 100 amino acids in length).