Functional Analysis Of Bifunctional Catalase-Peroxidase Gene MakatG1in Metarhizium Acridum

Metarhizium acridum is one of the most economically important insect pathogenicfungi and widely used as an insect biocontrol agent. It infects insects by directpenetration of the cuticle followed by multipication in the hemocoel and therefore, moreand more people are focus on the application of Metarhizium acridum. The infectionprocess involves conidia adherence to the insect cuticle, germination, formation ofappressorium, penetration of the cuticle, propagation in the hemocoel, finally causingdeath of the host. The cuticle is the first barrier to entomopathogenic fungi and therefore,the process before and during penetration plays a pivotal role in virulence. Base on thefull-length cDNA library constructed from M. acridum hyphae growing on locust wings,an EST that is the homolog of bifunctional catalase-peroxidase gene was chosen forfurther analysis and named MaKatG1. To explore the biological role of the MaKatG1inthe development and infection processes of M. acridum, we generated the disruptionmutant through homologous recombination.The main results are as follows:1. Molecular characteristics of MaKatG1.Based on the sequence of the entire M. acridum genome and the full-length cDNAlibrary from M. acridum hyphae growing on locust wings, a KatG homolog from M.acridum was cloned and named MaKatG1and the sequence was submitted to theGenBank database （accession numbers JN935023）. The full open reading frame waspredicted to encode a734amino-acid protein with a theoretical molecular weight of81kD （pI=5.88）, and did not contain a signal peptide. the MaKatG1gene product sharedsignificant homology with other KatGs.2. Targeted disruption of MaKatG1To explore the biological role of the MaKatG1in the development and infectionprocesses of M. acridum, we generated the disruption mutant through homologousrecombination of the MaKatG1ORF with a glufosinate-ammonium （GA） resistance（bar）.3. MaKatG1provides protection against H₂O₂and menadione.The plate sensitivity assays were used to validate the extent of twoâ–³katG1mutants and wild-type M. acridum resistance to H₂O₂and menadione. Growth of thewild-type and the twoâ–³katG1mutants was not significantly different5days post-inoculation on potato dextrose agar （PDA）. Growth of the wild-type was notsignificantly repressed on PDA medium supplemented with0.8mM,1.6mM,2.4mMH₂O₂, whereas growth of the twoâ–³katG1mutants was obviously repressed by H₂O₂.Furthermore,9days after inoculation, growth ofâ–³katG1mutants was also repressed byPDA medium supplemented with15μM and30μM menadione than the wild-type.H₂O₂killing assays were used to determine H₂O₂toxicity against M. acridum cells andthe potential of the MaKatG1gene product to detoxify H₂O₂. The LD50for theâ–³katG1mutant was1.8mM H₂O₂, which was significantly lower than that for the wild-type at6.3mM H₂O₂.4. MaKatG1gene has catalase and peroxidase activity.Greater catalase and peroxidase activities were identified in extracts fromwild-type compared with theâ–³katG1mutant. When exposed to oxidative conditions,the relative catalase and peroxidase activities of mutant reduced to about45%and60%compared to wild-type, respectively.5. MaKatG1contributes to virulence.The results revealed that the mutant was still to cause pathogen, but there was areduction in the rate and speed of mortality mediated by the mutant through topicalinfection. The LT50of the mutant （7.16±0.38days） was longer （P <0.01） than that ofthe wild-type （5.71±0.36days）. However, there was no significant difference betweenthe LT50of the wild-type and the mutant administered by injection, indicating thatMaKatG1affects virulence prior to entry of the fungus into the host. This was furtherconfirmed by microscopic observation of the decreased penetration of the knockoutmutant.6. MaKatG1contributes to penetration.The fungal growth in hemolymph of the infected locust was observed at12hintervals after injection or topical inoculation of conidia. There were no obviousdifferences in the number of hyphal bodies between the wild-type and mutant afterinjection. However, decreased hemolymph hyphal body concentrations were apparentfollowing topical application of the mutant strain as compared to the wild-type parent,suggesting a decreased penetration of the knockout mutant.7. MaKatG1affects the germination and appresorium formation on the locustcuticle.Germination and appressorium formation of the knockout mutant and the wild-typewere compared by inoculation of conidia onto the locust hind wings or plastic surfaces. On the locust wings, the percentage germination of the knockout mutant （43.3%） waslower （P <0.01） than the wild-type （51.8%）18h after inoculation. Furthermore, thewild-type produced more appressoria than the mutant24h post-inoculation （P <0.01）.However, there was no significant difference in germination and appressoriumformation between the wild-type and the knockout mutant on plastic surfaces. Theseobservations suggest that MaKatG1provides protection against ROS and otherantifungal compounds on the locust cuticle prior to gaining entry into the host.8. MaKatG1is related to UV-B tolerance.Following exposure to UV-B, the relative conidial germination rate of thewild-type was approximately83%and40%after1h and4h respectively. The knockoutmutant showed more sensitive than the wild type as exposure time increased, indicatingthat MaKatG1contributes to UV-B tolerance.