INVESTIGATION BZcon1, a SANT/Myb-Type Gene Involved in the Conidiation of Cochliobolus carbonum Jun-xiang Zhang,* Yi-xin Wu,† Honhing Ho,‡ Hao Zhang,† Peng-fei He,† and Yue-qiu He*,1 *Faculty of Plant Protection, and †Faculty of Agriculture and Biotechnology, Yunnan Agricultural University, Kunming 650201, Yunnan, China, and ‡Department of Biology, State University of New York, New Paltz, New York 12561 ABSTRACT The fungal pathogen Cochliobolus carbonum (anamorph, Bipolaris zeicola) causes Northern KEYWORDS Leaf Spot, leading to a ubiquitous and devastating foliar disease of corn in Yunnan Province, China. Asexual Bipolaris zeicola spores (conidia) play a major role in both epidemics and pathogenesis of Northern Leaf Spot, but the conidia molecular mechanism of conidiation in C. carbonum has remained elusive. Here, using a map-based cloning sporulation strategy, we cloned a single dominant gene, designated as BZcon1 (for Bipolaris zeicola conidiation), which clone encodes a predicted unknown protein containing 402 amino acids, with two common conserved SANT/ Northern Leaf Myb domains in N-terminal. The BZcon1 knockout mutant completely lost the capability to produce con- Spot idiophores 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. In many phytopathogenic fungi, asexual sporulation in the form of fungal disease by deciphering the molecular mechanism involved in conidia is critical in the life cycle, and so deciphering the molecular asexual sporulation of plant pathogenic fungi. mechanisms involved in conidiation development is a prerequisite to Asexual spores play a major role in the epidemics of Cochliobolus provide novel strategies for disease management. The survival of these carbonum (anamorph, Bipolaris zeicola), which causes Northern Leaf fungi is ensured by the production and dissemination of spores that Spot, a ubiquitous and devastating foliar disease of corn in many are long-lived and highly resistant to environmental stresses. Spore regions of the world (Nelson 1959, 1970; Welz and Leonard 1994; germination represents a critical stage in the life cycle of fungi and Zhang et al. 2013). In the wild, C. carbonum exists in the asexual form, constitutes a prerequisite for colonization in a new environment reproducing mainly through the production of conidia. As soon as the (Lafon et al. 2005). The process of conidiation involves many com- conidia are in contact with the corn surface, they release a copious, mon developmental themes, including gene expression, cell speciali- two-layered, extracellular matrix (ECM) resulting in the adhesion of zation, and intercellular communication. For example, in Aspergillus germlings of the fungus to the host surface (Evans et al. 1982; Apoga nidulans conidiation, three central regulatory genes (brlA, abaA,and et al. 2001). After penetration of the germ tubes, infectious hyphae wetA) control conidiation-specific gene expression and determine the initially grow within the plant cells. Eventually, the plant cells are order of gene activation during conidiophore development and co- killed and thousands of conidia could be released from each lesion. nidial maturation (Park and Yu 2012). These studies suggest that it is Despite the importance of conidia in pathogenic epidemics, little is important to develop new strategies to control the occurrence of known about the molecular mechanism of conidiation in C. carbonum. In ascomycetes, conidia formation requires the temporal and spatial control of cell differentiation, which is a process under Copyright © 2014 Zhang et al. polygenic control (Adams et al. 1998; Etxebeste et al. 2010). To date, doi: 10.1534/g3.114.012286 no genes involved in conidiation have been reported in the asexual Manuscript received April 6, 2014; accepted for publication May 31, 2014; development of C. carbonum. Therefore, it would be valuable to iden- published Early Online June 3, 2014. fi This is an open-access article distributed under the terms of the Creative tify and characterize genes that are speci cally involved in conidia Commons Attribution Unported License (http://creativecommons.org/licenses/ formation for an in-depth understanding of the asexual development. by/3.0/), which permits unrestricted use, distribution, and reproduction in any Development of genetic maps and the identification of molecular medium, provided the original work is properly cited. markers closely linked to target genes have allowed the map-based Supporting information is available online at http://www.g3journal.org/lookup/ fi suppl/doi:10.1534/g3.114.012286/-/DC1 cloning of speci city genes governing the phenotype (Sweigard et al. 1Corresponding author: Faculty of Plant Protection, Yunnan Agricultural University, 1995; Farman and Leong 1998; Ma et al. 2006). Here, via both a Kunming 650201, Yunnan, China. E-mail: [email protected] map-based cloning strategy and functional experiments, this study Volume 4 | August 2014 | 1445 characterizes a novel C. carbonum gene, BZcon1, which is a determi- Linkage marker screening of the conY locus nant of conidiation in this fungus. BZcon1 encodes a potential binding Pools of DNA for bulked segregant analysis (BSA) (Michelmore et al. protein containing 402 amino acids as putative transcription factors 1991) were prepared by combining equal amounts of DNA from five and regulates the conidiation in C. carbonum. conY or five conN progeny strains. RAPD (Welsh and McClelland 1990; Williams et al. 1990), ISSR (Zietkiewicz et al. 1994), and SRAP MATERIALS AND METHODS (Li and Quiros 2001) amplifications were performed on each pool and Fungal strains, cultures, and transformation parental strains. RAPD reaction productions were analyzed by elec- Field isolates S92 and S129 were collected from corn plants with trophoresis on agarose gels (1.2%). The specific fragment for conY Northern Leaf Spot symptoms in Yunnan Province, China, in 2010. pools was excised from the agarose gels. ISSR and SRAP reaction Experimental strains Aa82, Aa108, and Aa113 were obtained from the productions were resolved in polyacrylamide gels (6%) coupled with progeny of the cross S92 · S129. Other strains in this study are shown silver-staining. The exclusive band for conN pools was excised from in Table 1. Mating types of all isolates were determined by polymerase polyacrylamide gels as described by Zhang et al. (2009). The PCR chain reaction (PCR) amplification as described previously by Gafur production was excised and purified using the MinElute Gel Extrac- et al. (1997). Mycelia collected from potato dextrose agar (PDA) plates tion Kit (Sangon Biotech, China). The eluted fragment was ligated into were used for isolating fungal genomic DNA and RNA. Mycelia col- pMD18-T simple vector (Takara, Japan) following the supplier’s lected from Fries medium (Fries 1978) were used for obtaining the instructions and transformed into competent Escherichia coli TG1, protoplasts. Protoplasts were isolated and transformed as described by and then sequenced by Beijing Genomics Institute (BGI; Beijing, Liu and Friesen (2012). The selection medium was supplemented with China). The sequence obtained was used to design primers for se- 100 mg/ml hygromycin B (Roche, USA) or 500 mg/ml G418 (Amer- quence characterized amplified regions (SCARs) (Paran and Michelmore esco, USA) for the screening for hygromycin B–resistant (HR) or 1993) analysis. The successful SCAR primer sequences used are shown G418-resistant (GR) transformants, respectively. Single conidium of in Table 3. the transformant was isolated to ensure nuclear homogeneity (Choi et al. 1999). If lack of conidia precluded single-spore isolation, then Genetic mapping of the gene BZcon1 transformants were transferred three to four times to select against For genetic linkage analysis, SCAR amplifications were performed heterokaryons (Lev et al. 1999). Isolates and mutants were stored on with genomic DNA from 271 progenies of the cross between Aa113 filter paper disks as previously described (Silué et al. 1992). and S129. Segregation data obtained were analyzed with the software Mapmaker version 3 (Lander et al. 1987). Parameters for map con- Cross and conidiation assay struction were a minimum logarithm of odds (LOD) score of 3.0 and The isolates S92, Aa82, and Aa113 of C. carbonum produced abun- a maximum recombination fraction of 0.4. The Kosambi function was dant conidia (conY), whereas the isolate S129 did not produce conidia used to compute the recombination distances in centimorgans (cM). (conN) on PDA plates. Crossing was performed according to the de- scription of Welz and Leonard (1994) on Sachs agar medium (Gafur Construction and screening of BAC library et al. 1998). Pseudothecia were harvested 20 to 25 days after crossing, The Aa113 genomic DNA bacterial artificial chromosome (BAC) and single ascospores were isolated from the asci and allowed to library was constructed as described by Brosch et al. (1998) germinate on PDA. For conidiation assays, all isolates were incubated with minor modifications. After HindIII partial digestion, DNA frag- on PDA plates (6-cm diameter) at 25° for 10 d in the dark. Then, the ments 75 to 120 kb in size were ligated into plasmid pBeloBAC11 and colony surface was washed with 10 ml sterile distilled water (SDW) electroporated into E. coli TG1 using an electroporator (Eppendorf, and the conidia suspension was filtered through two layers of cheese- Germany). After electroporation, the cells were resuspended in 600 ml cloth. The amount of conidia per milliliter of the suspension was of LB medium (10 g tryptone, 5 g yeast extract, and 10 g NaCl in 1000 determined by counting the conidia with a hemacytometer using light ml deionized water), allowed to recover for 1 h at 37° with gentle microscopy. An isolate was regarded as a conY or a conN progeny with shaking, and then plated on LB agar containing chloramphenicol (12.5 the presence or absence of conidia, respectively. Each isolate was mg/ml). The plates were incubated overnight, and the recombinants tested at least four times.