1.8.45
CHARACTERIZATION OF A GENE ENCODING CYANIDE HYDRATASE IN LEPTOSPHAERIA MACULANS, THE CAUSAL AGENT OF BLACKLEG DISEASE OF OILSEED BRASSICAS

AC SEXTON and BJ HOWLETT

School of Botany, University of Melbourne, Parkville 3052, Australia

Background and objectives
Blackleg disease, caused by the ascomycete fungus Leptosphaeria maculans, results in significant losses to oilseed Brassica crops such as Brassica napus (canola) worldwide. In Australia, plant breeders are currently developing Indian mustard, B. juncea, as an alternative source of edible oil, and this species is more resistant to L. maculans than is canola. However, L. maculans isolates which can attack Indian mustard varieties have recently been found in Australia [1]. Indian mustard contains high levels of sulphur-containing glucosides called alkenyl glucosinolates, particularly 2-propenyl glucosinolate. When the plant is wounded, glucosinolates are hydrolysed by the plant enzyme myrosinase to release toxic compounds such as isothiocyanates (e.g. mustard gas), thiocyanates and nitriles, which are toxic to a wide range of organisms including the blackleg fungus. We are seeking L. maculans genes involved in detoxifying alkenyl glucosinolates or glucosinolate hydrolysis products. In this paper we describe the sequence and transcriptional regulation of a gene encoding a cyanide-detoxifying enzyme, cyanide hydratase. Cyanide hydratase catalyses the conversion of hydrogen cyanide, a breakdown product of nitriles, into the less toxic compound formamide.

Results and conclusions
A cDNA clone encoding cyanide hydratase was isolated from L. maculans using a probe (generously provided by Dr H. VanEtten, University of Arizona, USA) from another phytopathogenic fungus, Gloeocercospora sorghi. The L. maculans cyanide hydratase gene is present as a single copy, and the predicted amino-acid sequence has very high sequence similarity (76% identity) to the G. sorghi protein.

Cyanide hydratase in L. maculans is transcribed at a very low level in complete medium, at slightly higher levels in the presence of hydrolysis products of 2-propenyl glucosinolate (1.0 mM), and at extremely high levels after 4 h in the presence of potassium cyanide (0.1 and 1.0 mM), as shown by hybridization of the cDNA to an mRNA sized 1.1 kb. The cyanide hydratase mRNA was also detected during infection of Indian mustard cotyledons. Since cyanide hydratase transcription in L. maculans is inducible by 2-propenyl glucosinolate hydrolysis and much more strongly by potassium cyanide, this suggests that cyanide may be released during glucosinolate breakdown in Indian mustard. To test this we measured the amount of hydrogen cyanide released from macerated leaves of Indian mustard and canola, and from pure 2-propenyl glucosinolate hydrolysis. No cyanide was detected from pure 2-propenyl glucosinolate or from Indian mustard leaves, however a small amount (25 nmol/g fresh weight) was released from adult canola leaves. Canola has a high content of hydroxy aliphatic glucosinolates, which yield hydroxynitriles upon hydrolysis, and in other plants hydroxynitriles are known to breakdown into hydrogen cyanide. Therefore L. maculans may need to detoxify cyanide during infection of canola. Further work will involve testing whether the cyanide hydratase gene is expressed during infection of adult canola and Indian mustard plants, and disrupting the gene to test whether a lack of cyanide hydratase causes any reduction in fungal virulence.

References
1. Salisbury PA, Ballinger DJ, Wratten N et al., 1995. Australian Journal of Experimental Agriculture 35, 665-672.