This year, in our project, we constructed a new expression vector working effectively in the fungus and transformed into Metarhizium anisopliae 128 to increase its adhesion, penetration and immune-avoidance capacity. Therefore, we performed experiment on three aspects and the experimental characterizations of our parts are shown as follows.
In this part, a strong promoter, PgpdA allows the HsbA protein to be expressed without induction.
The HsbA( BBa_K2788000 ) from Beauveria bassiana encodes a kind of membrane surface hydrophobic protein which helps our spores adhere to the wax on the cockroach body surface. With the overexpression of HsbA, our spores can adhere to the cockroach more effectively . Then it will be followed as spore germination, formation of germinal tube and appressorium.
This part was insert into the expression vector by restriction sites EcoRI and PstI (Fig.1), and the correct construction of this recombinant plasmid was confirmed by PCR identification and sequencing of the PCR products.
We transformed the expression vectors into Metarhizium anisopliae 128 by CaCl ₂ -PEG induction method, and the positive clone was screened by G418 and colony PCR.
The transformed strain Metarhizium anisopliae 128 was grown in 1/4 SDAY liquid medium, and obtain total protein by FastPrep and ultrasonic crushing. The lysate was then centrifuged and the supernate were electrophoresed on a sodium dodecyl sulfate(SDS)-12% (wt/vol) polyacrylamide gel, followed by Coomassie blue staining.(Fig.3)
Besides, by using the method of comparing four areas of each wing in the scanning electron microscope before and after treatment which is ‘put the petri dishes on WD-9405B horizontal shaking table and shaked at the lowest speed for 10 minutes (to ensure the spores on the wings impact by the water flow equivalently)’ in the HsbA macro verification protocol, we can finally compared whether there was any change of the position and number of spores in the observing area. (illustrated with Fig.4 and Fig.5)
It’s obvious that it changes a lot（circle in red）of the position and number of spores in the observing area of wild-type Metarhizium anisopliae 128 experimental groups.
It’s obvious that there is almost no change except for the place (circled in red) in the position and number of spores in the observing area of Metarhizium anisopliae HsbA transformant experimental groups.
Finally, the following chart (Fig.6) can be obtained by statistical data of four areas in all experimental groups.
In conclusion, this result confirmed that Metarhizium anisopliae HsbA transformant certainly enhanced the capacity of adhesion.
In order to make Metarhizium anisopliae penetrate the cockroach's body wall more efficiently, we transferred Bbchit( BBa_K2788001 ), a gene from Beauveria bassiana ARSEF 2860, which encodes chitinase that can be secreted extracellularly. Chitin decomposes, thereby cockroach body wall destroys, finally fungus enters the hemolymph.(Fig.7)
We constructed a shuttle vector to transform Bbchit and the positive clones were screened by G418 and colony PCR.(Fig.8)
The crude enzyme solution was obtained by cell disruption using ultrasonic,centrifuged to gain supernatant, followed by SDS-PAGE protein electrophoresis and Coomassie blue staining.(Fig.9)
To determine the activity of chitinase, we improved a method based on the DNS colorimetric method(Kan Zhuo, Xiaozhen Shi). First, the standard curve was drawn with different concentration gradients of glucose solution.Second, took 0.5 ml culture solution of the wild-type and transformant grown for 1, 3, 5, 7, 9 and 12 days for enzyme activity test respectively,obtained the crude enzyme solution by filtering the culture solution. Then mixed the crude enzyme solution with 1% colloidal chitin, incubated at 37℃ for 60 minutes, and then added 0.5 mL DNS.Set in boiling water bath for 10 minutes. The absorbance of sample was measured by spectrophotometer and the enzyme activity was calculated according to the standard curve. There were three replicates of each group,perform three parallel experiments in each replicate, and the final data were averaged.
We calculated activity based on the standard curve formula: U=（A540+0.03279）/2.202 (Fig.10), a summary of the data at different times is made into a line chart as follows.We can see that after 12 days the transformant’s enzyme activity is still growing while the wild-type is falling.(Fig. 11)
In order to verify the function of Bbchit at the macroscopical level, we improved Kan Zhuo's chitin transparent circle method for verification. We stained the czapek solid medium without chitin colloids in red with 0.1% Congo red dye solution and then inoculated wild-type and transformed Metarhizium.Then compared the size of the colony and the transparent circle between wild type and transformant. The size measured is the diameter of the chitin transparent ring (R2) and colony(R1). The ratio of the diameters showed as R2/R1.The conclusion that the chitinase activity of Metarhizium anisopliae is enhanced.
Therefore, these results confirmed that the chitinase activity of Metarhizium anisopliae The enzyme activity of Bbchit transformant is about 1.3 times higher than wild-type's Metarhizium anisopliae 128. Our modified fungus certainly enhanced the ability of penetration.
Immune avoidance: MCL1
During evolution, insects have developed a very strong immune system against entomopathogenic fungi In hemolymph, hemocytes, and plasma play important roles in eliminating the fungi that are present in the hemolymph, But arm race between parasitic fungi and their insect hosts never stops. The fungus has mechanisms to overcome the immune systems of insects.,for example, the lower level of β-1,3-glucan in the cell surface does not stimulate the host immune response. We extract a gene named MCL1 from Metarhizium robertsiiARSEF 23, which encodes collagen-like protein to combine and block β-1,3,-glucan.This gene works like putting an “invisible cloak ”on the fungus so that immune avoidance can happen.
In this part, PgpdA is a strong promoter that allows MCL1 to be expressed without induction. of hemolymph.
We transferred the expression vector MCL1-pBC by CaCl ₂ -PEG induction method, then screen transformant by G418 resistance genes and colony PCR.PCR product was identified by agarose gel electrophoresis (Fig.14)
The transformed strain Metarhizium anisopliae 128 was grown in 1/4 SDAY liquid medium, and obtain total RNA by using RNAiso Plus(TAKARA), reverse transcription by using TAKARA PrimeScript™ RT reagent Kit, then perform quantitative PCR. (Fig.15)
We gain the total protein by FastPrep and ultrasonic crushing. The lysate was then centrifuged and the supernate was electrophoresed on a sodium dodecyl sulfate(SDS)-12% (wt/vol) polyacrylamide gel, followed by Coomassie blue staining.
In order to verify the ability of immune-avoidance of M.anasopliae,we inject hyphae homogenate into cockroaches as experimental groups and normal saline as control groups, then extract hemolymph in 0.5h, 1h , 2h, 4h, 8h, 12h and 24h. Meanwhile extract hemolymph from cockroaches that were not injected as group 0h. Count the nodules formed of hemocytes(Criteria: more than 10 hemocytes assemble closely)(Fig.17) and observe hemolymph smear under phase contrast microscope(Fig.18) <4>
Fig.18. Immune response of cockroaches’hemocytes to Metarhizium anisopliae spores under phase contrast microscope.A.B.C. hemolymph without injection. The number of hemocytes is relatively large and the shape is normal. Almost no nodule formation; D,G,J,M,P,S,V:control,almost no nodules formed; E:0.5h of WT injection,blood cells aggregated to form nodules,which means immune response on hypha;F: 0.5h of transformant injection,almost no nodule formed;H:1h of WT injection,more nodules was formed;I :1h of transformant injection, nodules started to form but was less than WT group;K:2h of WT injection , nodules were becoming larger;L:2h of transformant injection, nodules were smaller than WT group;N:4h of WT injection, nodules were less and smaller than 2h;O:4h of transformant injection, almost no nodules formed;Q:8h of WT injection, nodules were Most disintegrating.R:8h of transformant injection, Metarhizium anisopliae was growing in the shape of yeast;T:12h of WT injection, the number of hemocytes and nodules decreased; U:12h of transformant injection, blood cells were destroyed and cell debris can be seen;W:24h of WT injection, blood cells’ structure were destroyed, a large amount of cell debris cen be seen; X:24h of transformant injection, Metarhizium aerated in large numbers and few blood cells left. Wild-type M.anasoplise triggers more intense immune response than MCL1 transformant, while hemocytes degraded more in transformant groups.
In conclusion, MCL1 could trigger host immune response less and promote immune-avoidance.
In order to confirm the limited concentration of Tryptophan, we did a macro experiment. We put Metarhizium in an L-Tryptophan concentration gradient Petrie dishes from 0.05% to 0.14%. with solid and liquid czapek. After six days of cultivation, it can be seen in the
We can see the ten photos that, in the solid czapek, the Metarhizium can stay alive at the L-Trp concentration of 0.09% or higher while it could not grow well or die at a lower concentration. We can also see that Metarhizium could not survive without L-Tryptophan.
We put the Metarhizium in 100mL liquid czapek with different concentration L-Tryptophan, after 6 days of cultivation, here is the result:
We can see the chart and graph that, in the liquid czapek, the Dry Weight of Metarhizium stay in a stable level from 0% of [Trp] to 0.08% of [Trp], because it didn’t grow at this low concentration. At the L-Trp concentration of 0.09% or higher, its dry weight grow as the concentration of L-Tryptophan grow.
From our experiment we can see that, the limited Tryptophan concentration for Metarhizium to survive is 0.9% of Tryptophan.
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