Mol Cell Biol 18(2): 953-959

Viral Repression of Fungal Pheromone Precursor Gene Expression

Department of Plant Pathology and Microbiology, Texas A&M University, College Station, Texas 77843-2132

^{Corresponding author. Mailing address: Department of Plant Pathology and Microbiology, Texas A&M University, Room 120 L. F. Peterson Building, College Station, TX 77843-2132. Phone: (409) 845-8288. Fax: (409) 845-6483. E-mail:}ude.umat@neflanav.

^{Present address: Department of Internal Medicine and Biochemistry, Southwestern Medical School, Dallas, TX 75235-8573.}

^{Present address: Department of Cell Biology, Baylor College of Medicine, Houston, TX 77030.}

Received 1997 Mar 10; Revisions requested 1997 Apr 23; Accepted 1997 Jun 6.

Abstract

Biological control of chestnut blight caused by the filamentous ascomycete Cryphonectria parasitica can be achieved with a virus that infects this fungus. This hypovirus causes a perturbation of fungal development that results in low virulence (hypovirulence), poor asexual sporulation, and female infertility without affecting fungal growth in culture. At the molecular level, the virus is known to affect the transcription of a number of fungal genes. Two of these genes, Vir1 and Vir2, produce abundant transcripts in noninfected strains of the fungus, but the transcripts are not detectable in virus-infected strains. We report here that these two genes encode the pheromone precursors of the Mat-2 mating type of the fungus; consequently, these genes have been renamed Mf2/1 and Mf2/2. To determine if the virus affects the mating systems of both mating types of this fungus, the pheromone precursor gene, Mf1/1, of a Mat-1 strain was cloned and likewise was found to be repressed in virus-infected strains. The suppression of transcription of the pheromone precursor genes of this fungus could be the cause of the mating defect of infected strains of the fungus. Although published reports suggest that a Gα_i subunit may be involved in this regulation, our results do not support this hypothesis. The prepropheromone encoded by Mf1/1 is structurally similar to that of the prepro-p-factor of Schizosaccharomyces pombe. This is the first description of the complete set of pheromone precursor genes encoded by a filamentous ascomycete.

Abstract

Chestnut blight, caused by the ascomycete Cryphonectria parasitica, is one of the most devastating plant diseases in recorded history. Because of this disease, only occasional root sprouts grow where the American chestnut (Castanea dentata) was once dominant in eastern North America. A similar fate for the European chestnut (C. sativa) in European forests and orchards was prevented by a naturally occurring biological control of the fungus. The basis of this biological control is infection of C. parasitica by double-stranded RNA viruses which reduce the virulence of the fungus, causing a condition termed hypovirulence. The effect of the virus on reducing the virulence of this plant pathogen is the basis of an effective biological control of chestnut blight (1, 7, 13, 16, 24, 25).

One of these hypoviruses, CHV1-713, causes no detectable effects on the growth rate of its host in culture but perturbs normal developmental processes such as sporulation and virulence. The visible symptoms of CHV1-infected colonies growing on agar are a white rather than orange colony color and poor asexual sporulation, providing a useful phenotype for the study of this virus. The sexual cycle of the fungus is also perturbed by the virus, with the female parent being sterile. At the molecular level, the virus causes transcriptional down-regulation of a number of host genes (10). The cloning and identification of the function of some of these genes have provided us with a group of molecular markers of virus infection. We have previously reported the cloning of three of these genes: Crp, coding for an abundant hydrophobic protein located on the cell surface (28); Lac1, coding for an extracellular laccase (18); and Vir2, a gene identified as being necessary for sexual reproduction (27).

Vir2 and a related gene, Vir1, were identified as mRNAs found by subtractive hybridization between cDNAs of an uninfected strain of the fungus and the total RNA of an isogenic virus-infected strain (17). Vir2 was cloned and deleted; the Vir2 null mutant (dm18) exhibited a phenotype that partially mimicked virus symptoms of the host, i.e., reduced asexual sporulation and impaired sexual fertility. Attempts at sexual crosses of the null mutant produced barren perithecia. The Vir2 gene encodes a small protein of 23 amino acids with a C-terminal CAAX motif, a prenylation signal common to all known fungal lipopeptide pheromones (27). We report here that Vir2 encodes a mating-type specific pheromone precursor, and we describe the cloning of the other two pheromone genes of this fungus. Our results demonstrate that a filamentous ascomycete contains pheromone precursor genes encoding precursors with structural characteristics and maturation processing signals similar to those reported from the ascomyceteous yeasts. We also report that the virus represses the transcription of each of the fungal pheromone precursor genes of both mating types of C. parasitica, which could cause the mating defects exhibited by the virus-infected strains of the fungus.

ACKNOWLEDGMENTS

This work was supported by grants from the USDA National Research Initiative (90-37290-5671) and the National Science Foundation (MCB-9205818).

The technical assistance of Y.-B. Sun, D.-G. Tang, and G. Meyer is greatly appreciated, as is the advice and gift of strains from Kathy Borkovich.

ACKNOWLEDGMENTS

REFERENCES

References

1. Anagnostakis S LBiological control of chestnut blight. Science. 1982;215:466–471.[PubMed][Google Scholar]
2. Arie T, Christiansen S K, Yoder O C, Turgeon B GEfficient cloning of ascomycete mating type genes by PCR amplification of the conserved MAT HMG box. Fungal Genet Biol. 1997;21:118–130.[PubMed][Google Scholar]
3. Bolker M, Kahmann RSexual pheromones and mating responses in fungi. Plant Cell. 1993;5:1461–1469.[Google Scholar]
4. Carparelli, A., L. Zhang, D. Rigling, E. Gobbi, and N. K. Van Alfen. Unpublished data.
5. Chen B, Gao S, Choi G H, Nuss D LExtensive alteration of fungal gene transcript accumulation and elevation of G-protein-regulated cAMP levels by a virulence-attenuating hypovirus. Proc Natl Acad Sci USA. 1996;93:7996–8000.[Google Scholar]
6. Choi G H, Chen B, Nuss D LVirus-mediated or transgenic suppression of a G-protein α subunit and attenuation of fungal virulence. Proc Natl Acad Sci USA. 1995;92:305–309.[Google Scholar]
7. Gao S, Nuss D LDistinct roles for two G protein α subunits in fungal virulence, morphology, and reproduction revealed by targeted gene disruption. Proc Natl Acad Sci USA. 1996;93:14122–14127.[Google Scholar]
8. Heiniger U, Rigling DBiological control of chestnut blight in Europe. Annu Rev Phytopathol. 1994;32:581–99.[PubMed][Google Scholar]
9. Imai Y, Yamamoto MThe fission yeast mating pheromone P-factor: its molecular structure, gene structure, and ability to induce gene expression and G₁ arrest in the mating partner. Genes Dev. 1994;8:328–338.[PubMed][Google Scholar]
10. Jaynes R A, Elliston J EHypovirulent isolates of Endothia parasitica associated with large American chestnut trees. Plant Dis. 1982;66:769–772.[PubMed][Google Scholar]
11. Kazmierczak P, Pfeiffer P, Zhang L, Van Alfen N KTranscriptional repression of specific host genes by the mycovirus Cryphonectria hypovirus 1. J Virol. 1996;70:1137–1142.[Google Scholar]
12. Kazmierczak, P., P. McCabe, and N. K. Van Alfen. Unpublished data.
13. Larson T G, Choi G H, Nuss D LRegulatory pathways governing modulation of fungal gene expression by a virulence-attenuating mycovirus. EMBO J. 1992;11:4539–4548.[Google Scholar]
14. Larson T G, Nuss D LAltered transcriptional response to nutrient availability in hypovirus-infected chestnut blight fungus. EMBO J. 1994;13:5616–5623.[Google Scholar]
15. MacDonald W L, Fulbright D WBiological control of chestnut blight: use and limitations of transmissible hypovirulence. Plant Dis. 1991;75:656–661.[PubMed][Google Scholar]
16. Milgroom M GPopulation biology of the chestnut blight fungus, Cryphonectria parasitica. Can J Bot. 1995;73:S311–S319.[PubMed][Google Scholar]
17. Moore T D E, Edman J CThe α-mating type locus of Cryptococcus neoformans contains a peptide pheromone gene. Mol Cell Biol. 1993;13:1962–1970.[Google Scholar]
18. Nuss D LBiological control of chestnut blight: an example of virus-mediated attenuation of fungal pathogenesis. Microbiol Rev. 1992;56:561–576.[Google Scholar]
19. Powell W A, Van Alfen N KDifferential accumulation of poly(A)^{RNA between virulent and double-stranded RNA-induced hypovirulent strains of}Cryphonectria (Endothia) parasitica. Mol Cell Biol. 1987;7:3688–3693.[Google Scholar]
20. Rigling D, Van Alfen N KRegulation of laccase biosynthesis in the plant-pathogenic fungus Cryphonectria parasitica by double-stranded RNA. J Bacteriol. 1991;173:8000–8003.[Google Scholar]
21. Spellig T, Bolker M, Lottspeich F, Frank R W, Kahmann RPheromones trigger filamentous growth in Ustilago maydis. EMBO J. 1994;13:1620–1627.[Google Scholar]
22. Sprague G F., Jr Combinatorial associations of regulatory proteins and the control of cell type in yeast. Adv Genet. 1990;27:33–62.[PubMed]
23. Sprague G F, Jr, Thorner J W Pheromone response and signal transduction during the mating process of Saccharomyces cerevisiae. In: Jones E W, Pringle J R, Broach J R, editors. The molecular and cellular biology of the yeast Saccharomyces. Vol. 2. Cold Spring Harbor, N.Y: Cold Spring Harbor Laboratory Press; 1992. pp. 657–744. [PubMed]
24. Sweigard J A, Orbach M J, Valent B, Chumley F GA miniprep procedure for isolating genomic DNA from Magnaporthe grisea. Fungal Genet Newsl. 1990;31:44.[PubMed][Google Scholar]
25. Turner G E, Borkovich K AIdentification of a G protein α subunit from Neurospora crassa that is a member of the G_i family. J Biol Chem. 1993;268:14805–14811.[PubMed][Google Scholar]
26. Van Alfen N K Hypovirulence of Endothia (Cryphnectria) parasitica and Rhizoctonia solani. In: Buck K W, editor. Fungal virology. Boca Raton, Fla: CRC Press, Inc.; 1986. pp. 143–162. [PubMed][Google Scholar]
27. Van Alfen N K, Jaynes R A, Anagnostakis S L, Day P RChestnut blight: biological control by transmissible hypovirulence in Endothia parasitica. Science. 1975;189:890–891.[PubMed][Google Scholar]
28. Wang Z, Brown D DA gene expression screen. Proc Natl Acad Sci USA. 1991;88:11505–11509.[Google Scholar]
29. Zhang L, Churchill A C L, Kazmierczak P, Kim D, Van Alfen N KHypovirulence-associated traits, induced by a mycovirus of Cryphonectria parasitica, mimicked by target inactivation of a host gene. Mol Cell Biol. 1993;13:7782–7793.[Google Scholar]
30. Zhang L, Villalon D, Sun Y, Kazmierczak P, Van Alfen N KViral-associated down-regulation of the gene encoding cryparin, an abundant hydrophobic protein from the filamentous fungus, Cryphonectria parasitica. Gene. 1994;139:59–64.[PubMed][Google Scholar]