Agrobacterium-mediated T-DNA Insertional Mutagenesis And Identification Of The Morphological And Metabolic Mutants In Trichoderma Reesei

Trichoderma reesei (anamorph: Hypocrea jecorina) is a biotechnically important filamentous fungus known as an efficient producer of enzymes and proteins. As one of the best known cellulolytic organisms, T. reesei can produce readily and in large quantities a complete set of extracellular cellulases for the degradation of cellulose and hemicellulose. Because of its capacity to secrete enzymes in high yields, T. reesei has been exploited as an industrial host for homologous and heterologous protein production. Knowledge gained from the genetic sequence of T. reesei will give researchers important insights into the synthesis and regulation of important enzymes, which will accelerate the development of bioprocesses for a range of industries and applications. The genome sequence of T. reesei, as the first member of Trichoderma species, has been completed and available freely (http://genome.jgi-psf.org/Trire2/Trire2.home.html). This work has significantly advanced the understanding of this biotechnically important organism and offered an opportunity to identify important new genes critical to its growth and development. Therefore, developing high-through-put methods to work out the biological function of these genes is of great significance. Genetic approaches, producing a large mutant population by disruption or replacement of genes and then studying the related fungal phenotypes, are powerful tools for deciphering gene function.1. Agrobacterium-mediated transformation (AMT) of T. reesei as an efficient tool for random insertional mutagenesisIn this study, filamentous fungus T. reesei QM9414 was successfully transformed with Agrobacterium tumefaciens AGL-1 for random integration of transforming DNA (T-DNA). The T-DNA binary vector pBI-hph was constructed by introducing the hygromycin B phosphotransferase gene (hph) isolated from pAN7-1 into pBIN121 between the right and left borders of T-DNA. Co-cultivation of T. reesei conidia or protoplasts with A. tumefaciens containing pBI-hph in the presence of acetosyringone resulted in the formation of hygromycin B-resistant fungal colonies with high transformation frequency. Nine randomly selected resistant clones were proved to be stable through mitotic cell division. The integration of the hph gene into T. reesei genome was determined by PCR and Dot blot analysis. TAIL-PCR successfully rescued the T-DNA borders and the flanking genome sequences from the putative transformants. Sequence analysis of the nine recombination junctions from TAIL-PCR revealed that no homology was found among them. The results showed that T-DNA inserts occurred evidently by fusing DNA at T-DNA borders via random recombination. It suggests that Agrobacterium-mediated transformation is a potentially powerful tool towards tagged mutagenesis and gene transfer technology for T. reesei. A population of T-DNA tagged T. reesei mutants has been obtained from the AMT transformation.2. Screening and characterization of the morphological and developmental mutants in T.reeseiWe screened the T-DNA tagged insertional lines of T. reesei and found seven strains with abnormal phenotypes. Among these strains, two mutants had T-DNA insertion at the TrCCD1 gene locus with different locations, designated ccdO and ccdP, respectively. ccdO had a T-DNA insertion into the open reading frame of the gene TrCCD1 and ccdP had a insertional mutagenesis in the promoter region of the same gene. Sequence analysis showed that TrCCD1 gene encodes a carotenoid cleavage dioxygenase, which belongs to RPE65 family found in fungi and higher organisms. Disruption of TrCCD1 caused several dramatic phenotypes. The hyphae from wild-type QM9414 colony extend rapidly while those from the disruptants radiate slowly and tend to form an incompact colony. The mutants are particularly distinctive in producing slow-growing hyphae and the colony growth rates of the mutants and wild-type strains were compared on minimal agar over a period of several days. The colony growth rate of each mutant strain was almost uniform; however, it was only one-half to three-fifth that of the parent, QM9414. When the strains grew on the MM with 0.2% Triton X-100, colonies from disruptants form some long aerial mycelia, in contrast to wild type. ccd mutants are also defective in sporulation. Though colonies from disruptants form some long aerial mycelia, they are difficult to form conidiophores. Wild type QM9414 strain produced green spores dispersing the Petri dishes, and the ccdO and ccdP disrupts only displayed a bit green in the center of medium after four days of incubation. On the same time, color displayed in mutants' medium was quite different from that in wild-type's medium, and carotenoid content in the former medium was higher than that in the later. These results suggeset that CCDs are important for the hyphal growth and conidiophore development of filamentous fungi.The other five mutants selected from phenotype screening were: PM2, PM48, HP7, HP13 and HPL1. They displayed different morphology. In contrast to the wild type, their hyphae extended slowly, some of them had radiant colony or white colony, and even some couldn't perform sporulation. Most interestingly, PM2 had curved hyphal tips and their mycelium showed many small "heads". TAIL-PCR successfully rescued the T-DNA borders and the flanking genome sequences from the mutants PM48 and HPL1. In PM48, T-DNA was inserted into the ORF of the gene encoding a cell cycle-associated protein. While in HPL1, the gene disrupted by T-DNA insertion probably encoded a polyadenylate-binding protein, and the mutation of this gene reduced the cellulolytic ability of T. reesei.3. Screening and characterization of the cellulolytic mutants in T.reeseiAbout two hundred T. reesei AMT transformants, which had been proved to be inserted by T-DNA on the molecular level, were screened by the sizes of the clearing zones on cellulose CF11 agar plates. Among them, four mutants showed different cellulolytic ability: PM3, PM23, HPL1 and 36H-6. The clearing zones of PM3 and PM23 were about 30% bigger than that of QM9414, while HPL1 and 36H-6 had the smaller zones, and even 36H-6 couldn't form the complete clearing zone. The T. reesei FPA activity reached the maximal value between 18h and 24h. The rnaximal FPA activities of PM3 and PM23 were both 30% higher than that of wild type, and PM3 reached the maximal activity ahead of the wild type and PM23 had the longer high FPA activity. Another two mutants HPL1 and 36H-6 had the lower FPA activity than that of QM9414, and even HPL1 had only 1/3 activity of QM9414 at the maximal value. The T. reesei CMCase activity reached the maximal value at 24h. The maximal CMCase activities of PM3 and PM23 were both higher than that of wild type, while HPL1 and 36H-6 had the lower CMCase activity. It is noticeable that the FPA and CMCase activites of HPL1 always keep the lower level. In HPL1, the gene disrupted by T-DNA insertion probably encoded a polyadenylate-binding protein, which involved in RNA processing and modification.4. Construction of the binary vector containing GFP reporter gene and transformation of AgrobacteriumTwo other binary vectors containing both GFP and HPH as double-selective markers based on different Ti plasmids were constructed. In order to construct double-selective-marker vector, an intermediate plasmid pBI-gfp was obtained from the ligation of four fragments, which contained GFP expression cassette with GFP gene joined to the trpC terminator and controlled by the gpdA promoter from A. nidulans. Then this cassette was isolated from pBI-gfp and inserted into pBI-hph between the RB and LB of T-DNA. Thus, the first double-selective-marker binary vector pBI-gh came into being. After co-cultivation between A. tumefaciens and T. reesei, the HPH-resistance transformants was selected. PCR analysis established that the gfp and hph gene also was incorporated into the genome, but we were not able to confirm GFP fluorescence in an examination of the selected transformants. With the available data, we cannot draw conclusions about the expression of this transgene in T. reesei but note that it has been problematic in other organisms owing to aberrant mRNA processing and codon preference. Another binary vector, designated pCB-hg, consisted of a pCAMBIA1301 backbone containing the hph and gfp genes. This GFP gene was isolated from pIG1783 plasmid, which had successfully expressed GFP in different filamentous fungi. pCB-hg vector had been introduced into A. tumefaciens. The AMT of T. reesei and the selection of the transformants are undergoing experimental procedures.