The Photoreceptor Components FaWC1 and FaWC2 of Fusarium asiaticum Cooperatively Regulate Light Responses but Play Independent Roles in Virulence Expression

Abstract

Fusarium asiaticum belongs to one of the phylogenetical subgroups of the F. graminearum species complex and is epidemically predominant in the East Asia area. The life cycle of F. asiaticum is significantly regulated by light. In this study, the fungal blue light receptor white collar complex (WCC), including FaWC1 and FaWC2, were characterized in F. asiaticum. The knockout mutants ΔFawc1 and ΔFawc2 were generated by replacing the target genes via homologous recombination events. The two mutants showed similar defects in light-induced carotenoid biosynthesis, UV-C resistance, sexual fruiting body development, and the expression of the light-responsive marker genes, while in contrast, all these light responses were characteristics in wild-type (WT) and their complementation strains, indicating that FaWC1 and FaWC2 are involved in the light sensing of F. asiaticum. Unexpectedly, however, the functions of Fawc1 and Fawc2 diverged in regulating virulence, as the ΔFawc1 was avirulent to the tested host plant materials, but ΔFawc2 was equivalent to WT in virulence. Moreover, functional analysis of FaWC1 by partial disruption revealed that its light-oxygen-voltage (LOV) domain was required for light sensing but dispensable for virulence, and its Zinc-finger domain was required for virulence expression but not for light signal transduction. Collectively, these results suggest that the conserved fungal blue light receptor WCC not only endows F. asiaticum with light-sensing ability to achieve adaptation to environment, but it also regulates virulence expression by the individual component FaWC1 in a light-independent manner, and the latter function opens a way for investigating the pathogenicity mechanisms of this important crop disease agent.

Keywords: Fusarium asiaticum; White collar complex; photobiology; transcription factor; virulence.

Figure 1

Identification of two photoreceptor genes, Fawc1 and Fawc2, in F. asiaticum. (A). Schematic demonstration of the domains of WC1 and WC2 orthologs from N. crassa, F. graminearum, and F. asiaticum. Accessions of the amino acid sequences are as follows: WC1 (NCU02356); WC2 (NCU00902); FgWC1 (FGSG_07941); FgWC2 (FGSG_00710); FaWC1 (KX905081.1); FaWC2 (MT019868). Domains of these photoreceptor proteins were analyzed via the SMART online tool (http://smart.embl-heidelberg.de/). PAS: Per-period circadian protein; Arnt: Ah receptor nuclear translocator protein; Sim: Single-minded protein, NLS: Nuclear Location Singal, Zn: Zinc finger binding to DNA consensus sequence. (B). Transcript levels of Fawc1 and Fawc2 are regulated by light. The horizontal axis indicates light treatment time. The bars present mean values ± SD of three replicate samples.

Figure 2

Generation of the transgenic mutants of F. asiaticum. Schematic diagrams of homologous recombination occurred between the replacement vector carrying the hygromycin resistance marker (hph) and the target gene (Fawc1) of F. asiaticum strain EXAP-08, resulting in the knockout mutant (∆Fawc1). Meanwhile, the wild-type Fawc1 or its truncated versions, Fawc1^∆LOV and Fawc1^∆Zn, were amplified from genomic DNA of EXAP-08 and cloned into the flu6 plasmid, and then transformed into the ∆Fawc1 mutant, resulting in the complementation strain ∆Fawc1-C, and ∆Fawc1-C^∆LOV and ∆Fawc1-C^∆ZnF mutant strains. The knockout mutant ∆Fawc2 and its complementation strain ∆Fawc1-C were generated via a similar strategy.

Figure 3

Effect of light on UV-C resistance. (A). Serial dilutions of all strains were point-inoculated onto complete agar medium (CM). After the UV irradiation of indicated dosages, the plates were incubated for one day in light (left) or darkness (right). (B). The relative expression level of the deduced photolyase gene Faphr1 in wild-type and mutant strains as influenced by light. DD, samples cultured for 48 h in darkness; LL, samples experienced 47 h culture in darkness followed by one hour of light illumination. The bars present mean values ± SD of three replicate samples.

Figure 4

Effect of light on carotenogenesis. (A). Carotenoid pigment accumulation of the tested strains cultured in liquid CM for four days under constant light (LL) or darkness (DD). (B). Measurement of carotenoid contents in the mycelium of each strain harvested from the liquid shaking culture in (A). (C) and (D). Relative expression levels of deduced carotenoid biosynthesis genes CarRA and CarB in wild-type and mutant strains under light (LL) or darkness (DD). The bars in B, C, and D present mean values ± SD of three replicate samples.

Figure 5

Regulation of Fawc1 and Fawc2 on the sexual reproduction development processes of F. asiaticum. The perithecium formed by each strain on carrot agar medium was observed via stereomicroscope. Via picking up the perithecia and pressing them on glass slides for microscopic analysis, mature ascospores were observed from the perithecia of EXAP-08, ∆Fawc1-C, ∆Fawc1-C^∆ZnF, and ∆Fawc2-C strains, while in contrast, only mycelium biomass could be found in the immature perithecium of ∆Fawc1, ∆Fawc2, and ∆Fawc1-C^∆LOV mutant strains.

Figure 6

Virulence assay on wheat confirms that Fawc1 regulate the virulence of F. asiaticum in a light-independent manner. (A). Wheat coleoptiles were inoculated with 5 µL conidial suspensions and were kept humid inside a plastic box. Fungal strains for test include the wild-type EXAP-08, ∆Fawc1, ∆Fawc1-C, ∆Fawc1-C^∆LOV, ∆Fawc1-C^∆ZnF, ∆Fawc2, and ∆Fawc2-C. Photographs were taken at four days post inoculation. (B). Statistical analysis of lesion sizes caused by each fungal genotype. Different letters represent a significant difference at p < 0.05. The bars present mean values ± SD (n = 20).