Functional Analysis Of Arginase Gene MaAGA In Metarhizium Acridum

Metarhizium spp. is a model species for studying the fungal entomopathogenic mechanism. It has also been employed as a fungal insecticide and widely applied in the biological control of pests. Metarhizium acridum shows host specificity towards locust, and has been successfully employed in the locust control in Asia, Africa and Australia. M. acridum spreads and initiates insect infection in the form of conidium, which is a principal factor causing natural epidemic entomopathogenic disease. Therefore, to improve M. acridum as a fungal insecticide, much efforts should been put on developing strains with better sporulation ability.M. acridum produces conidia by either normal conidiation or microcycle condiation. During normal conidiation, conidia are produced upon differentiated stalk cells separated from mycelia. With respect to microcyle conidiation, the differentiation of mycelia to form conidia is bypassed, and conidia are produced shortly after conidial germination in a mass yeast-like slime forms. Compared to normal condiation, microcycle conidiation produces much more spores with better heat tolerance. With the attempt to improve the sporulation ability of M. acridum, it is necessary to elucidate the molecular regulation mechanism concerning microcycle condiation.The arginine metabolism pathway was reported to be involved in conidiation in the filamentous fungi Aspergillus nidulans and Coniothyrium minitans. The contribution of arginine metabolism to sporulation, however, has not been characterized in the entomopathogenic fungi including M. acridum. In this paper, we carried out the functional analysis of the M. acridum gene encoding arginase during microcycle conidiation, which is a key enzyme in arginine catabolism catalyzing the hydrolysis of arginine into orthinine and urea. Base on the full-length cDNA library constructed from M. acridum hyphae growing on locust wings, an EST that is the M. acridum gene encoding arginase was chosen for further analysis and named MaAGA. To explore the biological role of the MaAGA, we generated the disruption mutant through homologous recombination.The main results obtained in our study are listed as follows: (1) Based on the sequence of the entire M. acridum genome and the full-length cDNA library from M. acridum hyphae growing on locust wings, the full-length gene MaAGA encoding arginase was cloned and sequenced (GenBank accession numbers HM068369). The ORF (open reading frame), which was978bp long and interrupted by two introns, was predicted to encode a325amino-acid with a theoretical molecular weight of35.05kD (pI=5.1). The predicted protein contained arginine-specific binding domain and other arginase-conserved amino acid residues, two manganese ion binging domain. In addition, the MaAGA gene is conserved, whose homology were21-23%compared with plant,30-38%compared with bacterium and40-53%and40-49%compared with fungal and animal arginases, respectively.(2) When grown on the normal conidiation agar medium1/4SDA, the expression of M. acridum gene MaAGA was relatively higher at the1st day than that on the microcycle condiation medium SYA, and the expression decreased afterwards and maintained at a steady state between the2nd and12th day, but was lower than that on the SYA between the2nd and8th day. When grown on the medium SYA, the expression of MaAGA decreased between the1st day and2nd day, but increased between the2nd and6th day, and decreased again afterwards and maintained at a steady state between the8nd and12th day.(3) The arginase gene MaAGA targeted deletion mutants were generated by homologous recombination. Compared to the wide type strain, the mutant⊿Maaga is incapable of utilizing arginine as the sole nitrogen source. However, the mutant could use the arginine hydrolyzing products orthinine and urea as efficiently as the wide type strain. When grown in the SYA agar medium, the vegetative hyphal growth of the wide type strain was enhanced by the exogenously added arginine, ornithine or urea. The growth of the mutant⊿Maaga in the presence of the arginine was comparatively less than that of the wide type strain, due to the inability of the mutant⊿Maaga in catabolizing arginine. The growth of the mutant⊿Maaga in the presence of the ornithine or urea was similar to that of the wide type strain. However, the exogenously added ornithine or urea could not restore the growth of the mutant⊿Maaga to the level of that of the wide type strain.(4) When grown on either the agar medium1/4SDA or SYA, the exogenous addition of arginine reduced the conidiation ability of M. acridum. Besides, the extent of reduction in conidiation was dependent on the concentration of arginine.(5) The mutant⊿Maaga could grow normally as the wide type strain on1/4SDA medium supplemented with different stress chemicals. However, the mutant⊿Maaga produced abundant white hyphal growth on SYA medium containing different stress chemicals.(6) Microscopic observation revealed that the microcycle conidiation processes were similar between the wide type strain and the mutant⊿Maaga within40h of inoculation. After60h of inoculation, the wide type strain continued to produce abudant conidia, but the deletion mutant⊿Maaga switched into vegetative hyphal growth with producing few conidia.(7) The mutant⊿Maaga showed higher germination percentage than the wide type strain in the early stages of germination when grown on the1/4SDA or SYA agar medium. However, there was no significant difference between the wide type strain and the deletion mutants after10h germination. The tolerance of M. acridum to.UV exposion was not affected in the mutant⊿Maaga, but the tolerance to wet heat was significantly increased in the mutant⊿Maaga compared to the wide type strain.