Functional analysis of the glutamate transporter gene Cgglt1 from Colletotrichum gloeosporioides on the mango

Abstract：【Objective】Colletotrichum gloeosporioides (Penz.) Penz. & Sacc is the main causal agent of anthracnose in mangos (Mangifera indica) , which is very prevalent in the field and postharvest storage stage, and has great economic impact on the mango industry worldwide. Mango anthracnose is one of the main diseases in all mango planting countries, usually causing mango leaf spot, flower wilt, fruit soft rot, and directly decreases its fruit yield and commercial value. China is one of the main growing areas of mango; in 2014, the cultivated areas reached 17 320 hm2, and the total yield reached 143.77 million ton, which was ranked eighth in the world. A large amount of fungicides are used to control mango anthracnose every year, but it still causes significant economic losses. So it is necessary to develop more effective control measures. Studies of the infection mechanism of C. gloeosporioides on mango can help to improve our knowledge and develop some new control targets. It has been reported that C.gloeosporioides could alkalinize the host tissue during disease development. The pH modulation is not a host-mediated effect, but rather it is pathogen dependent. Host tissue alkalinization results from active secretion of ammonia which is induced by relatively low pH values. The glt1 is a nitrogen metabolism related gene, which regulates the synthesis and metabolism of ammonia. In our previous work, a glutamate transporter gene, named Cgglt1, was cloned from C. gloeosporioides isolated from diseased mango leaves. In order to identify the role of the glutamate transporter gene Cgglt1 in the infection process, particularly, to reveal the relationship between virulence and the gene, the Cgglt1 is disrupted and the phenotypes of the mutant are analyzed in the present study.【Methods】Resulting 5'and 3'Cgglt1 fragments and gfp-hyg B gene are inserted into p UC19 via T-cloning site using an In-Fusion®HD Cloning Kit (Clontech, Mountain View, CA, USA) , and then transferred into Escherichia coli DH5α. So a knockout vector is constructed by replacing the Cgglt1 coding region with the hyg B gene and s GFP gene from p GH33. The positive clones are sequenced and the Cgglt1 gene replacement vector with the right sequence is stored and named p Cgglt1 GH1, in which the gfp-hyg B gene is flanked by Cgglt1 fragments. The protoplasts of C. gloeosporioides are prepared using the lysozyme degrading method. The knockout fragment GLTGH amplified from p Cgglt1 GH1 is transformed into C. gloeosporioides protoplasts using the PEG4000-mediated method. Hygromycin-resistant transformants are screened and purified on hygromycin-containing SR plates and confirmed by applying PCR methods. Finally, by performing a series of comparative phenotype analysis, including culture morphology, mycelial growth, conidia production, germination and appressorium formation, suitable pH value ranges and pathogenicity on mango leaves, are performed between the obtained knockout mutant △ Cgglt1 and its wild-type.【Results】The Cgglt1 gene knockout vector p Cgglt1 GH1 was successfully constructed. The mutant △Cgglt1 was obtained and confirmed by transforming the knockout fragment glt1 GH into the C. gloeosporioides protoplasts and PCR detection. Phenotypic analysis showed that the colony of mutant △Cgglt1 became light in color and was always white, and grew slower in terms of the radial growth rate than with the blackish-brown wild-type. The mutant △Cgglt1 failed to produce conidia on the PDA, even after a prolonged incubation of 12 days, and only produced 103 spores per mL in PD broth or seldom generated spores when inoculated on detached wounded leaves with mycelial plugs. The conidial germination ratio of mutant △Cgglt1 significantly accelerated in the presence of free water but could not form the appressoria on moist glass slides. The wild-type produced a large number of conidia by macroscopic observation in PD broth and on detached wounded leaves. The ends of the wild-type germ tubes swelled and formed melanized appressoria after incubation for 4 h. This indicated that the mutants lose their ability to form infection structure appressorium. Mycelial adaptability of the mutant to ambient pH changes in terms of radial growth rates and the most suitable pH value ranges changs from 7-9 for wild types to 6-8 for the mutants, indicating that mycelia growth prefers relative low pH. When slightly wounded leaves were inoculated with wild-type mycelial plugs, the lesions were visible 2 days after inoculation, and became typical anthracnose spots with an average lesion diameter around 21.7 mm (on bronze-colored tender leaves) and 21.0 mm (on light green leaves) 5 days after inoculation. However, △Cgglt1 caused smaller necrotic spots than the wild-type on slightly wounded leaves, with necrotic spot size of 18.8 mm (on bronze-colored tender leaves) and 18.7 mm (on light green leaves) 5 days after inoculation. When unwounded leaves were inoculated with wild-type mycelial plugs, the lesions were visible 2 days after inoculation, then enlarged to form typical anthracnose necrotic spots of 19.7 mm (on bronze-colored tender leaves) and 17.9 mm (on light green leaves) 5 days after inoculation. In contrast, no lesions emerged on unwounded leaves inoculated with △Cgglt1 mycelial plugs or control agar plugs5 days after inoculation. This suggests that the knockout of the Cgglt1 gene results in the loss of the ability to directly penetrate into the mango leaf epidermal surface as well as reduce its expansion efficiency in leaf tissue.【Conclusion】The glutamate transporter gene Cgglt1 of C. gloeosporioides on mangos was involved in colony pigmentation and mycelial growth, conidia production, conidia germination rate, appressorial formation, adaptability to relative alkalic pH, and virulence to mango leaves. The glutamate transporter gene glt has been cloned in several other pathogenic fungi, but its function was onlyidentified in C. gloeosporioides on avocados. The knockout of the glt1 gene resulted in only significant reductions in appressorium formation and pathogenicity on avocados. This indicates that the functions of glt1 genes are significantly different in C. gloeosporioides from different hosts.