Cloning of Insertion Site Flanking Sequence and Construction of Transfer DNA Insert Mutant Library in Stylosanthes Colletotrichum

Abstract

Stylosanthes sp. is the most important forage legume in tropical areas worldwide. Stylosanthes anthracnose, which is mainly caused by Colletotrichum gloeosporioides, is a globally severe disease in stylo production. Little progress has been made in anthracnose molecular pathogenesis research. In this study, Agrobacterium tumefaciens-mediated transformation was used to transform Stylosanthes colletotrichum strain CH008. The major factors of the genetic transformation system of S. colletotrichum were optimized as follows: A. tumefaciens’ AGL-1 concentration (OD600), 0.8; concentration of Colletotrichum conidium, 1×106 conidia/mL; acetosyringone concentration, 100 mmol/L; induction time, 6 h; co-culture temperature, 25°C; and co-culture time, 3 d. Thus, the transformation efficiency was increased to 300–400 transformants per 106 conidia. Based on the optimized system, a mutant library containing 4616 mutants was constructed, from which some mutants were randomly selected for analysis. Results show that the mutants were single copies that could be stably inherited. The growth rate, spore amount, spore germination rate, and appressorium formation rate in some mutants were significantly different from those in the wild-type strain. We then selected the most appropriate method for the preliminary screening and re-screening of each mutant’s pathogenic defects. We selected 1230 transformants, and obtained 23 strains with pathogenic defects, namely, 18 strains with reduced pathogenicity and five strains with lost pathogenicity. Thermal asymmetric interlaced PCR was used to identify the transfer DNA (T-DNA) integration site in the mutant that was coded 2430, and a sequence of 476 bp was obtained. The flanking sequence of T-DNA was compared with the Colletotrichum genome by BLAST, and a sequence of 401 bp was found in Contig464 of the Colletotrichum genome. By predicting the function of the flanking sequence, we discovered that T-DNA insertion in the promoter region of the putative gene had 79% homology with the aspartate aminotransferase gene in Magnaporthe oryzae (XP_003719674.1).

Genome‐wide identification of pathogenicity, conidiation and colony sectorization genes in Metarhizium robertsii

“…The identification of important pathogenicity genes (Zhao et al ., ; Fang et al ., ) and a new fungal pigmentation gene cluster (in this study), further confirm the utility of the library for identifying genes involved in important biological processes. However, results with DNA insertion library projects (Jeon et al ., ; Chen et al ., ; Michielse et al ., ), require follow up experiments to validate associations of phenotypes of the mutants with the disrupted genes.…”

“…Studies with plant pathogenic fungi such as Magnorporthe oryzae , Fusarium oxysporum and Stylosanthes colletotrichum have shown that a large DNA insertion mutant library is a very useful tool for identifying pathogenicity loci (Jeon et al ., ; Chen et al ., ; Chen et al ., ; Michielse et al ., ). We developed an efficient Agrobacterium ‐mediated transformation method for M. robertsii (Fang et al ., ), and used it to construct a T‐DNA insertion library with more than 20,000 transformants.…”

Transcriptomic and Physiological Analysis of the Effects of Exogenous Phloretin and Pterostilbene on Resistance Responses of Stylosanthes against Anthracnose

“…Colletotrichum gloeosporioides is a hemibiotrophic fungal pathogen which causes lesions on leaves, stems, fruits and other parts of woody or herbaceous plant [ 4 , 5 , 6 ]. Anthracnose caused by C. gloeosporioides is one of the most destructive and predominant diseases of stylo worldwide, greatly limiting the yield of stylo [ 7 ]. Typical symptoms of stylo anthracnose are spreading irregular lesions in leaves with dark margins and light brown centers, which spread to all tissues during the late stage of the disease [ 8 , 9 ].…”

Development of an efficient vector system for gene knock-out and near in-cis gene complementation in the sugarcane smut fungus

“…However, the released DNA fragment flanked by the T-DNA arms inserts into the host genome in a random manner 24 – 26 . Indeed, it is based on this feature, AMT has been used to generate mutant library for microbes 27 , 28 .…”