The Saccharomyces cerevisiae chitinase, encoded by the CTS1-2 gene, as an antifungal and biocontrol agent

[摘要] ENGLISH ABSTRACT:Fungi are an extremely diverse group of organisms and, by acting as pathogens, they cancolonise various other organisms, including humans, plants and animals. The effect of this isusually detrimental, not only to agricultural crops and livestock, but also to human well-being.The extensive farming of crops and livestock requires persistent control of fungal populations,commonly through the use of chemical fungicides. However, the exclusive use of fungicides isno longer a sustainable practice, as a result of serious problems, such as increasing fungicideresistance in pathogen strains, the high costs of fungicides, as well as concern about theenvironment. The search by producers and scientists for alternative control measures is anongoing process.The fungal cell wall consists of polysaccharides that not only playa role in protection of thefungi, but also in relaying signals for the invasion and infection of susceptible hosts. Chitin, apolysaccharide composed of N-acteylglucosamine (GleNAc) residues linked by P-1,4 glucosidiclinkages, is one of the major components of the fungal cell wall, where it plays an important rolein the apical growth of the vegetative hyphae.Chitinases (EC 3.2.1.14) are abundant proteins produced by a variety of microorganismsand plants and are necessary for the hydrolysis of the chitin polymer. During the invasion ofmany plant species by a pathogen, the production of a specific group of proteins, designatedpathogenesis-related (PR) proteins that include chitinases, is induced as part of their defenceresponse. Due to the facts that pathogenic fungi contain chitin in their cell walls and that plantchitinases are induced upon pathogen attack, chitinases have been confirmed as an integral andcrucial part of the plant's natural defence response. Chitinases have increasingly been targetedto upregulate plants' endogenous disease resistance mechanisms through transgenicoverexpression in a variety of hosts.Several species of fungi, including various Trichoderma spp., are potent biocontrol agentsof plant pathogenic fungi and insects. The antagonistic activities of these biological controlagents towards phytopathogens are based on the secretion of extracellular hydrolytic enzymes,such as cell wall-degrading chitinase enzymes. However, biological control is not restricted tonaturally occurring biocontrol agents. Through the process of genetic transformation, otherfungal or yeast species can be enhanced to produce their own chitinases or other antimicrobialsubstances more effectively in order to yield potent biocontrol agents.Various types of chitinases have been applied in the production of fungal resistant plantsand some research has been done on the application of chitinases, from a variety ofmicroorganisms, as biological control agents. In contrast, very little is known about theantifungal activity of the Saccharomyces cerevisiae chitinase enzyme, encoded by the CTS1-2gene. The CTS1-2 gene was utilised in this study as a candidate for overexpression in bothyeast and plant expression systems to analyse the ability of the encoding chitinase to inhibitfungal growth.The first objective of this study involved the high level expression and optimisation of thesecretion of the CTS1-2 gene in S. cerevisiae to render recombinant yeast with enhancedantifungal abilities and with possible applications as a biocontrol agent to control plantpathogenic fungi. It was hypothesised that high-level expression and efficient secretion wouldbe prerequisites in a biocontrol yeast strain. To this end, two strong promoters and terminatorswere included in the study and the secretion of the chitinase gene was evaluated by testingthree different secretion signals. The secretion signals included: the native CTS1-2 secretionsignal, the S. cerevisiae mating pheromone a-factor (MFa1) secretion signal, as well as theTrichoderma reesei f3-xylanase 2 (XYN2) secretion signal. The phosphoglycerate kinase 1(PGK1) and alcohol dehydrogenase 2 (ADH2) promoters and terminators were employed toachieve high-level expression.The results obtained from the analysis of the recombinant yeasts showed that the PGK1promoter-terminator constructs yielded high level CTS1-2-expressing and chitinase-producingstrains of S. cerevisiae PRY488. The ability of the different secretion signals to efficientlysecrete the overexpressed chitinase was analysed and it was found that the non-nativesecretion signals delivered significantly more protein to the extracellular environment. It wasthus evident that the performance of the MFa1 and XYN2 secretion signals was superior to thatof the native secretion signal. The antifungal activities of the recombinant chitinases producedby these constructs were tested in in vitro assays against Botrytis cinerea. The enzymes led toa significant reduction in hyphal development, caused by extreme structural damage to thehyphal tips, the hyphal cell walls as well as the ability of the fungus to form reproductive andsurvival structures, thereby confirming the antifungal abilities of this enzyme. The ADH2promoter-terminator constructs yielded CTS1-2 transcripts, but no chitinase activity could bedetected with any of these strains. The reasons for this still remain unclear.The second objective of this study was to assess the potential of the yeast chitinase gene toupregulate defence against fungal infection in planta. In order to elucidate this, the CTS1-2gene was constitutively overexpressed in tobacco plants, targeting the chitinase both to theintra- and the extracellular environment. The results obtained showed that the transgenictobacco lines regenerated in this study stably integrated the transgene, exhibiting transgeneexpression as well as the production of a biologically active yeast chitinase enzyme. The F,progeny were rigorously tested for resistance to B. cinerea, and both in vitro and in plantaassays confirmed that the yeast chitinase increased the plant's tolerance to fungal infection;some of the lines showed disease resistance of 65 and 70%. The plants expressing anextracellularly targeted chitinase gene are still under evaluation. Interesting results are expectedrelating to the effect of the chitinase on the plant surface with regards to disease resistance tofungal pathogens.In conclusion, the combined set of results from both the yeast and plant overexpressionstudies has confirmed the strong antifungal effect of yeast chitinases. The yeast CTS1-2chitinase could be instrumental in the development of a new generation of yeast strains withimproved antifungal capabilities. This enzyme could also play an important role in genetictransformation technologies aimed at enhanced disease resistance.

[发布机构] Stellenbosch University