The role of swnR gene on the biosynthesis pathway of the swainsonine in Metarhizium anisopliae

doi:10.21203/rs.2.23703/v1

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The role of swnR gene on the biosynthesis pathway of the swainsonine in Metarhizium anisopliae

https://doi.org/10.21203/rs.2.23703/v1

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Background: Swainsonine (SW) is the principal toxic ingredient of locoweeds, and is produced by fungi including Metarhizium anisopliae, Slafractonia leguminicola, and Alternaria oxytropis (found in locoweeds of Oxytropis). Studies of the SW biosynthesis pathway in these fungi have demonstrated the requirement for a swnK gene and the presence of a variety of other SWN cluster genes, but have not determined a precise role for the swnR gene, which encodes a NADB Rossmann-fold reductase, nor if it is necessary for the biosynthesis of SW. In this study, we used homologous recombination (HR) to knock out the swnR gene of M. anisopliae to determine its effect on the SW biosynthesis pathway.

Results: The concentration of SW was measured in the fermentation broth of M. anisopliae at 1 d, 3 d, 5 d and 7 d using a Q Exactive Mass Spectrometer. The gene for swnR was detected by RT-qPCR. To determine the role of the swnR gene in the SW biosynthesis pathway of M. anisopliae, we used PEG-mediated homologous recombination (HR) to transform a wild-type strain (WT) with a Benomyl (ben)-resistant fragment to knock out the swnR gene producing a mutant-type strain (MT). A complemented-type (CT) strain was produced by adding a complementation vector that contains the glufosinate (herbicide) resistance (bar) gene as a marker. The content of SW decreased, but was not eliminated in the fermentation broth of the MT strain, and returned to the original level in the CT strain.

Conclusions: These results indicate that the swnR gene plays a crucial role in the SW biosynthesis pathway of M. anisopliae, but suggests that another gene in the fungus may share the function of swnR.

Applied & Industrial Microbiology

General Microbiology

Swainsonine

swnR

M. anisopliae

mutant strain

complemented strain

Metarhizium anisopliae is an entomopathogenic fungus that can infect a variety of agricultural pest insects and produces swainsonine [1-3]. Swainsonine is an indolizidine alkaloid that inhibits alpha-mannosidase. Swainsonine is produced by a variety of fungi and is the major toxic component of locoweed plants, which are widely found in western China and North America [4-9]. Swainsonine (SW) causes locoism in grazing animals [10-12], when consumed for an extended time. SW also has anti-tumor properties and may enhance the body's immunity [13-16].

Recently, Cook et al. [17] sequenced the genome of two swainsonine-producing fungi Metarhizium robertsii, and compared their swainsonine producing genes to those in M. robertsii and other swainsonine-producing fungi. All the swainsonine-producing fungi of Metarhizium sp. contained a common gene cluster "SWN", which included swnH₁, swnH₂, swnK, swnN, swnR, swnA and swnT. Fungi that do not produce swainsonine do not contain the SWN cluster. These genes encode catalytic enzymes involved in the SW biosynthesis. swnN and swnR both encode NADB Rossmann-fold reductases. After annotating the "SWN" gene cluster, Cook et al. [17] successfully constructed a swnK knockout vector for the entomopathogen M. robertsii, and obtained a swnK mutant strain by HR. The LC-MS analysis showed that SW was eliminated in the fermentation broth of the mutant strain and the content of SW returned to normal after complementation with the swnK gene, indicating that the swnK gene is essential for SW biosynthesis.

The precise roles for the other genes in the SWN cluster were not determined. The aims of this study were to investigate the role of the swnR gene in the SW biosynthesis pathway of M. anisopliae. To accomplish this, swnR of M. anisopliae was knocked out using HR. The deletion of swnR led to a significant decrease in the SW content in the fermentation broth of M. anisopliae. The content of SW in the complemented strain was similar to that found for the wild type strain.

Detection of SW in fermentation broth of M. anisopliaein different periods

To detect the SW content in the fermentation broth at different time points, the same volume of M. anisopliae fermentation broth was concentrated, and the level of SW was detected using a Q Exactive Mass Spectrometer. The retention times for the SW peak of the SW standard and the SW test samples was 4.4 (Additional file 1: Figure S1A, S1B, S1C, S1D, and S1E). The standard curve was drawn according to the calculated regression equation: Y=49236.2＋12706.9X (R²=0.999) (Additional file 1: Figure S1F). From the linear regression equation of mass concentration-peak area of SW, the SW content in the fermentation broth of M. anisopliae was calculated to be 169.67 ± 50.78 μg/mg (Additional file 1: Figure S1A), 174.01 ± 45.79 μg/mg (Figure S1B), 116.72 ± 45.74 μg/mg (Figure S1C), and 104.85 ± 40.35 μg/mg (Additional file 1: Figure S1D), at 1, 3, 5, and 7 days of growth, respectively. Day 3 of fermentation gave the highest content of SW (Fig. 1).

RT-qPCR analysis of key catalytic enzyme genes in the SW biosynthesis pathway of M. anisopliae

RT-qPCR was conducted for genes in the SW biosynthesis pathway to determine their relative expression at 1d, 3d, 5d, and 7d. The expression of swnN was down-regulated from 1d to 3d. The expression levels of swnT and swnK decreased at 3 d and 5 d, and the expression of swnH₁ and swnH₂ did not significantly change. The expression of swnR gene was significantly up-regulated at 3 d and decreased at 5d and 7d (Fig. 2).

Screening for optimal conditions for protoplast preparation

We explored the conditions affecting the preparation of protoplasts of M. anisopliae to enhance genetic manipulation of the fungus. Hyphae produced from fermentation times of 1 d, 2 d, 3 d, 4 d, and 5 d were enzymatically digested for 3 h at 30 °C, 100 rpm. The optimal fermentation time of 2 d was determined based on protoplast yield (Fig. 3A). The effects of different enzymatic hydrolysis combinations and enzymatic concentrations were compared on protoplast preparations of M. anisopliae. Enzymatic hydrolysis using 1% snail enzyme, 1% cellulase, and 1% lysing enzymes was the best and produced the largest number of protoplasts (Fig. 3B). Duration of enzymatic hydrolysis time is also an important variable of the protoplast preparation process, so the optimal enzymatic hydrolysis time was assessed. When enzymatically hydrolyzed for 3 h, the hyphal wall was completely dissolved, releasing large numbers of protoplasts (Fig. 3C and 3D).

Production of MT and CT

To determine the role of the swnR gene in the SW biosynthesis pathway of M. anisopliae, homologous recombination was used to knock out swnR. The resulting transformant (1 out of 55) grew on SDA media containing 200 μg/mL benomyl. Subsequently, the L1/R1 primer set was used to identify the genomic DNA of the transformant (MT) using electrophoresis and sequencing (Fig. 4 and Additional file 3: Text S1). To verify the status of MT, a complement was produced by transforming the wild-type swnR gene in pBARGPE1 into the MT. The complement transformant was grown on SDA medium containing 2 mg/mL glufosinate and was identified as above.

Phenotypic observation and growth rate determination of WT, MT, and CT

The WT, MT, and CT isolates were grown on three SDA plates each with two colonies per plate for 3 d, 5 d, and 10 d (Fig. 5A) to compare growth. The colony diameters, phenotypes, and growth rates did not change significantly (Fig. 5B).

Q Exactive Mass Spectrometer detection of SW in fermentation broth of MT, CT and WT

The same volume of MT, CT and WT fermentation broth was concentrated, and the concentration of SW was detected using a Q Exactive Mass Spectrometer. The peak time in the test samples and the SW standard was 4.4 (Additional file 1: Figure S2A, S2B and S2C). The standard curve was drawn according to the calculated regression equation: Y=31302.5X-45910.5 (R²=0.997) (Additional file 1: Figure S2D). From the linear regression equation of mass concentration-peak area of SW, the content of SW in the fermentation broth of M. anisopliae was calculated to be 82.91 ± 15.92 μg/mg for WT (Additional file 1: Figure S2A), 56.42 ± 10.82 μg/mg for CT (Additional file 1: Figure S2C) and 5.71± 2.23 μg/mg for MT (Additional file 1: Figure S2B). The content of SW in the fermentation broth of the MT was significantly lower than that in the complemented strain and WT (Fig. 6).

While Cook et al. [17] demonstrated that the swnK gene is required for the SW biosynthesis of M. robertsii, no studies had determined which other genes are required for SW biosynthesis in M. anisopliae. We carried out a time course of SW in the fermentation broth of M. anisopliae, and compared the SWN gene expression for the same time course. The results showed that SW concentration was the highest at 3 d and the swnR gene was highly expressed at this time. This suggested that the swnR gene may play an important role in the SW biosynthesis pathway of M. anisopliae.

To further confirm the role of the swnR gene in the SW biosynthesis pathway of M. anisopliae, the swnR gene was knocked out using the benomyl resistance gene as a screening marker. The content of SW in MT was significantly reduced. Cook et al. [17] found that the swnK MT did not produce SW at all. However, in this study, after the swnR gene was knocked out, the content of SW was reduced, rather than completely absent. The cause of the low SW content may be due to the presence of a catalytic enzyme gene having the same function as the swnR gene. Since swnN is also a Rossman fold reductase, and is present in all SW-producing fungi, this could provide a similar activity and thus explain why SW was decreased rather than eliminated.

To demonstrate that the decrease in the content of SW was caused by the knock out of the swnR gene, the gene was complemented by inserting the wild-type swnR gene into the MT. The content of SW in the complement returned to normal levels, as predicted. Cook et al. [17] showed an increase in the content of SW after complementing the swnK gene, likely due to the high expression of the swnK gene in the complemented strain. We performed phenotypic observations and growth rate measurements on the MT, and found that it did not differ significantly from the wild-type strain. Therefore, the deletion of the swnR gene resulted in a decrease in the content of SW, but did not affect the radial growth of M. anisopliae. It is not known if sporulation or some other growth component was changed with the knockout.

The factors affecting the preparation of protoplasts and regeneration of filamentous fungi include the age of fungi, the choice of medium and enzymatic hydrolysate, enzymatic hydrolysis conditions and length of enzymatic hydrolysis [18,19]. We found that M. anisopliae grown on SDA medium for 3d produced the most protoplasts. The cell wall composition of M. anisopliae is reported to be very complex [20], which might have favored the compound enzyme for the release of the M. anisopliae protoplasts This study showed that the protoplast release of M. anisopliae was the highest when digested with 1% snail enzyme, 1% cellulose, and 1% lysing enzymes for 3 h.

SW can cause neurotoxicity in grazing animals [21-23] and seriously threatens the production and development of animal husbandry [8, 9, 24]. SW also has significant anticancer and antitumor effects [25-27]. However, the limited source of SW, the difficulty of artificial synthesis, low extraction efficiency and high market prices have greatly limited the development of SW for anticancer and anti-tumor applications [14, 28, 29]. The production of SW by microbial fermentation, specifically by M. anisopliae or S. leguminicola [30] might be possible if the SWN pathway is better characterized.

In this study, we demonstrated that the deletion of the swnR gene resulted in a decrease in swainsonine concentration in the fermentation broth of M. anisopliae and the recovery of swainsonine concentration to normal levels in the complemented strain. This suggests that the swnR gene plays an important role in the swainsonine biosynthesis pathway of M. anisopliae (Fig. 7). This study provides a preliminary research basis for the in-depth study of the swainsonine biosynthesis pathway and related catalytic enzyme genes.

Strain and fermentation culture

Metarhizium anisopliae (BNCC114445), obtained from Research Institude of Forest Ecology, Environment and Protection, Chinese Academy of Forestry, Beijing, China, was inoculated onto Sabouraud medium (SDAcontaining 50 µg/mL chloramphenicol and cultured at 30 °C for 10 days. Six 5 mm plugs of mycelium were transferred into 200 mL SDA liquid culture media and fermented at 28 °C, 180 rpm, for 1 d, 3 d, 5 d, or 7 d. The mycelium and the fermentation broth were filtered through three layers of Miracloth (EMD Millipore Corp, Billerica, MA, USA) to collect the mycelium. No conidia were detected from the cultures. The mycelium was dried at room temperature and stored at 4 °C for use.

RT-qPCR analysis of genes in the SW biosynthesis pathway of M. anisopliae

Fungal RNA was extracted using the E.Z.N.A. Fungal RNA Kit (Omega). RNA was reverse transcribed into DNA by using PrimeScript^TMRT reagent Kit (Takara). Primers for amplification of SwnN, SwnT, SwnK, SwnH1, SwnH2, SwnR, and 18S rRNA are shown in Table S1 of supporting information. All of the genes were amplified using RT-qPCR with the following conditions: 1 cycle of 95 °C for 10 min; 40 cycles of denaturation at 95 °C for 10 s, annealing at 55 °C for 90 s, and an extension at 72 °C for 32 s. Cultures were tested using RT-qPCR at 1 d, 3 d, 5 d, and 7 d.

Identification of swnR gene of M. anisopliae

Fungal DNA was extracted using the CTAB method. Primers for amplification of swnR were designed from the swnR sequence from Cook et al. [17], GeneBankKID61009 of M. anisopliae ARSEF 549. The swnR gene (GeneBank MN892388) was amplified from M. anisopliae DNA using L1/R1 primers(Additional file 2: Table S2).

Vector construction

The upstream and downstream fragments of the swnR gene (Additional file 1: Figure S3) and the benomyl (fungicide) resistance gene (ben) (Additional file 1: Figure S4) were inserted into pUC19 (Takara) digested with EcoR I/BamH I (Takara) (Additional file 1: Figure S5) using the In-Fusion® HD Cloning System (Takara) to construct a knockout construct targeting the swnR gene (Additional file 1: Figures S6 and S7 and Additional file 3: Text S1).

The primers L3 and R3 (Additional file 2: Table S2) were used to amplify the ben resistance gene from pBARGPE1-BenA (Wuhan Jingxiu Scientific Biotechnology Co., Ltd., China) as a template. The primers L2/R2 and L4/R4 (Table S1, supporting information) were used to amplify the upstream target fragment (swnR-I) and the downstream target fragment (swnR-II), respectively, of the swnR gene from the genomic DNA of M. anisopliae. The swnR-I, ben, swnR-II and the double-cut pUC19 vector were ligated using In-Fusion cloning. The swnR gene fragment was amplified using primers L1/R1 (Additional file 2: Table S2) from the genomic DNA of M. anisopliae. To produce a complementation vector, the swnR was inserted between trpC promoter and trpC terminator of pBARGPE1 vector, which contains the glufosinate (herbicide) resistance (bar) gene as a marker, using In-Fusion cloning (Fig. 4).

Preparation of protoplasts

Six 5 mm plugs from 10-day cultures of M. anisopliae grown on SDA media were transferred into each 200 mL flask of SDA liquid culture media, and incubated at 28 °C, 180 rpm for 1 d, 2 d, 3 d, 4 d and 5 d. The resulting mycelia were filtered through sterile miracloth. To the collected hyphae were added different concentrations of enzymatic hydrolysate (Sigma Aldrich) prepared with 1.2 M KCl, and hydrolyzed at 30 °C, 100 rpm, for 1 h, 3 h, 5 h, 7 h, and 10 h. The optimal combination of enzymes and conditions were determined based on protoplast yield. Yield from different enzymes, including 1% snail enzyme, 1% cellulase, and 1% lysing enzymes, and combinations of the enzymes were also tested. The enzymatically digested mixtures were filtered through a layer of sterile miracloth and two layers of filter paper into a sterile 50 mL centrifuge tube, and the protoplasts were washed extensively with 1.2 M KCl and centrifuged at 4000 rpm for 6 min at room temperature. After discarding the supernatant, 10 mL of STC Buffer (0.6 M Sorbitol; 10 mM Tris-HCl; 10 mM CaCl₂, pH 6.5) was added and the protoplasts were gently resuspended. The mixture was centrifuged at 4000 rpm for 6 minutes. After discarding the supernatant, 1 mL of STC Buffer was added. The protoplasts were then centrifuged at room temperature at 3500 rpm for 6 min, which was repeated. Finally, protoplasts were adjusted to 2-5×10⁷/mL for subsequent experiments.

PEG mediated DNA transformation

Transformation of the protoplasts were done as in Proctor et al. [32] Approximately 5-10 µg of the linearized swnR knockout vector was added to a 50 mL centrifuge tube containing 2-5×10⁷/mL protoplasts, and allowed to stand at room temperature for 20 min without shaking. Then 1-1.25 mL of 40% PTC (40% PEG 8000, 20% sucrose, 50 mM CaCl₂, 10 mM Tris-HCl) was added to the tube (mixed thoroughly by inversion), and let stand at room temperature for 20 min without shaking. Thereafter, 5 mL of TB₃ (0.3% Yeast Extract, 0.3% acid hydrolyzed casein, 20% sucrose) containing 50 g/mL ampicillin (Sigma Aldrich) was added and shaken at room temperature overnight. The overnight protoplasts were centrifuged at 4000 rpm for 6 min, the supernatant was discarded, and about 1 mL of the remaining liquid was used to suspend the remainder. The regenerated protoplasts were added to 10 mL of Bottom Agar (0.3% Yeast Extract, 0.3% acid hydrolyzed casein, 20% sucrose, 1% Agar) containing 100 µg/mL benomyl (Additional file 1: Figure S6). After incubation at 30 °C for 10 hours, Top Agar (0.3% Yeast Extract, 0.3% acid hydrolyzed casein, 20% sucrose, 1.5% Agar) containing 200 µg/mL benomyl was added. After 3-5 d, a single colony transformant grew on the plate, which was transferred to SDA medium containing 200 µg/mL benomyl. The wild type M. anisopliae was used as a control. The swnR gene mutant strain of M. anisopliae was named MT. The transformation of the complement vector was the same as described above, and 2 mg/mL of glufosinate (Additional file 1: Figure S8) was used for screening of the complement (CT).

PCR identification of MT and CT

The M. anisopliae strain carrying the benomyl resistance gene was used as a template, and PCR amplification was carried out using primer L1/R1. Subsequently, the complemented strain was subjected to the same PCR amplification using primers L1/R1 (Additional file 1: Table S2).

Phenotypic observation and growth rate determination of MT, CT and WT

Colonies of MT and WT of the same size were inoculated into the same position on the SDA medium and grown at 28 °C for 3 d, 5 d and 10 d, after which they were measured for diameter and photographed.

SW content detection of fermentation broth of WT, MT, CT in M. anisopliae

The WT, MT, CT strains were inoculated into SDA medium containing 50 µg/ml chloramphenicol and cultured at 28 °C for 10 days. Then six 5 mm plugs of each strain were transferred into 200 mL flasks of SDA (without agar) culture medium and grown at 28 °C, 180 rpm, for 3 d. The flasks of fermentation broth of WT, MT, and CT were combined by strain and filtered to obtain 500 mL of fermentation broth. The SW (control from Sigma Aldrich) in each extract was analyzed using Q Exactive Mass Spectrometer (Thermo Fisher) using the methods of Song et al. [33]. SW concentration was tested three times for each strain.

Statistical analysis

In this study, each measurement was tested three times. Statistical analysis was performed on the measured data using SPSS 20.0 software. The results were expressed as mean ± SEM. One-way ANOVA was performed on each sample, *P<0.05, indicating a significant difference between the two groups, **P<0.01, indicating that the difference between the two groups is highly significant. Results from the cultures were used for determining the optimal time periods for swainsonine production. The mass concentration peak area for SW was compared using linear regression. The colony diameters were measured by ruler. RT-qPCT data were analyzed using the 2 ^-△△CT method.

Ethics approval and consent to participate

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Availability of data and materials

All data generated or analyzed during this study are included in this published article [and its supplementary information files].

Competing interests

The authors declare that they have no competing interests.

Funding

This work was supported by the grants from the National Natural Science Foundation of China (No. 31201958) and the Key project of Natural Science Foundation of Shaanxi province (No. 2017JZ004) and the Science and Technology Special Fund Aid to Qinghai province (No. 2020-QY-210).

Authors’ contributions

H.L., B.Y.Z. and Q.Y.G. contributed to the conception of the focus for the study. L.S., R.J.S., Y.Z., Y.L.Z., B.L, Y.L.L. and W.X.Z. performed the experiments. J.L.W. and C.H.M. analyzed the data. L.S. and R.J.S. contributed to the compilation of all sections, figure and table design, and wrote the first draft of the manuscript. All authors contributed to revision, read and approved the submitted version of the manuscript.

Acknowledgements

Not Applicable.

Hino M, Nakayama O, Tsurumi Y, Adachi K, Shibata T, Terano H, Kohsaka M, Aoki H, Imanaka H. Studies of an immunomodulator, swainsonine. I. Enhancement of immune response by swainsonine in vitro. J. Antibiot. 1985;38:926-35. doi: org/10.7164/antibiotics.38.926.
Tian T, Li R, Lu X, Zhang L, Dong J, Zhang Z, Zhang Y. Screening for high virulence strains of Metarhizium anisopliae against Locusta migratoria manilensis from Baoding. Plant. Prot. 2009;5:65-69. doi: 10.3969/ j.issn.0529-1542.2009.05.015.
Roberts DW, Leger RJS. Metarhizium spp., cosmopolitan insect-pathogenic fungi: mycological aspects. Adv. Appl. Microbiol. 2004;54:1-70. doi: org/10.1016/S0065-2164(04)54001-7.
Huang Y, Zhang E, Pan W. Current status of locoweed toxicity. Shandong Sci. 2001;16:34-9.
Li J, Lu L, Cang Y, Dai C, Qiao J, Chen K. Study on the sterols of Hericium erinaceus extracts. Chinese J. Biochem. Pharmateutics. 2001;3:124-6.
Zhao B, Tong D, Ge P, Dong Q, Mo C. Locoweed harm investigation in the west grassland of China. Chinese J. Grassland. 2003;25:65-8.
Chen J, Ma R, Lu H, Wang Z, Zhao B. Progress on locoweed-fungal endophyte. Prog. Vet. Med. 2011;32:77-81. doi: org/10.1093/mp/ssq070.
Pryor BM, Creamer R, Shoemaker RA, McClain-Romero J, Hambleton S. Undifilum, a new genus for endophytic Embellisia oxytropis and parasitic Helminthosporium bornmuelleri on legumes. Botany. 2009;87:178-194. doi: org/10.1007/s00299-005-0913-4.
Baucom D, Romero M, Belfon R, Creamer R. Two new species of Undifilum, the swainsonine producing fungal endophyte, from Astragalus species of locoweed in the United States. Botany. 2012;90:866-875. doi: org/10.1139/b2012-056.
Panter KE, James LF, Stegelmeier BL, Ralphs MH, Pfister JA. Locoweeds: effects on reproduction in livestock. J. Nat. Toxins. 1999;8:53-62.
Wu C, Wang W, Liu X, Ma F, Cao D, Yang X, Wang S, Geng P, Lu H, Zhao B. Pathogenesis and preventive treatment for animal disease due to locoweed poisoning. Environ. Toxicol. Pharmacol. 2014;37:336-47. doi: org/10.1016/j.etap.2013.11.013.
Zhao B, Liu Z, Lu H, Wang Z, Sun L, Wan X, Guo X, Zhao Y, Wang J, Shi Z. Damage and Control of Poisonous Weeds in Western Grassland of China. Agri. Sci. in China. 2010;9:1512-1521.
White SL, Nagai T, Akiyama SK, Reeves EJ, Grzegorzewski K, Olden K. Swainsonine stimulation of the proliferation and colony forming activity of murine bone marrow. Cancer Commun. 1991;3:83-91. doi: org/10.1007/BF00841258.
Klein JL, Roberts JD, George MD, Kurtzberg J, Breton P, Chermann JC, Olden K. Swainsonine protects both murine and human haematopoietic systems from chemotherapeutic toxicity. Br. J. Cancer. 1999;80:87-95. doi: org/10.1038/sj.bjc.6690326.
Rooprai HK, Kandanearatchi A, Maidment SL, Christidou M, Trillo-Pazos G, Dexter DT, Rucklidge GJ, Widmer W, Pilkington GJ. Evaluation of the effects of swainsonine, captopril, tangeretinand nobiletin on the biological behaviour of brain tumour cells in vitro. Neuropathol. Appl. Neurobiol. 2001;27:29-39. doi: org/10.1046/j.0305-1846.2000.00298.x.
Hamaguchi J, Nakagawa H, Takahashi M, Kudo T, Kamiyama N, Sun B, Oshima T, Sato Y, Deguchi K, Todo S, Nishimura S. Swainsonine reduces 5-fluorouracil tolerance in the multistage resistance of colorectal cancer cell lines. Mol. Cancer 2007;6:58. doi: org/10.1186/1476-4598-6-58.
Cook D, Donzell B, Creamer R, Baucom DL, Gardner DR, Pan J, Moore N, Krasnoff SB, Jaromczyk JW, Schardl CL. Swainsonine Biosynthesis Genes in Diverse Symbiotic and Pathogenic Fungi. G3 (Bethesda) 2017;7:1791-1797. doi: org/10.1534/g3.117.041384.
Yu J, Keller N. Regulation of secondary metabolism in filamentous fungi. Annu. Rev. Phytopathol. 2005;43:437-458. doi: org/10.1146/annurev.phyto.43.040204.140214.
Abello J, Kelemu S, Garcia C. Agrobacterium-mediated transformation of the endophytic fungus Acremonium implicatum associated with Brachiaria grasses. Mycol. Res. 2008;112:407-413. doi: org/10.1016/j.mycres.2007.10.008.
Ranga V, Saini GK. Biochemical and molecular characterization of wild-type and fused protoplasts of Beauveria bassiana and Metarhizium anisopliae. Folia. Microbiol. (Praha) 2011;56:289-295. doi: org/10.1007/s12223-011-0055-8.
Marsh CD. The locoweed disease of the plains. United States Department of Agriculture, Washington, DC,1909.
Gardiner MR, Linto AC, Aplin THE. Toxicity of Swainsona canescens for sheep in western Australia. Australian J. Agri. Res. 1969;20:87-97. doi: org/10.1071/AR9690087.
Lu H, Cao DD, Ma F, Wang SS, Yang XW, Wang WL, Wang QW, Zhao BY. Characterisation of locoweeds and their effect on livestock production in the western rangelands of China: a review. Rangeland J. 2014;36:121-131. doi: org/10.1071/RJ13105.
Yu Y, Zhao Q, Wang J, Wang J, Wang Y, Song Y, Geng G, Li Q. Swainsonine-producing fungal endophyte from major locoweed species in China. Toxicon. 2010;56:330-338. doi: org/10.3892/mmr.2011.622.
Zou HXF, Zhang Z, Sun X. Research progress of a promising anti-cancer drug swainsonine. Chin. Tradit. Drugs 1997;28:437-439.
Liu B, Liu Y, Yin H, Chen X. Inhibition effect of swainsonine on the growth and metastasis of gastric cancer in vivo. Chin. J. Oncol. 1999;20:168-170.
Shi Y, Li M, Xu F. A review of the study on swainsonine--toxic elements of locoweed. Zhong Yao Cai. 1999;22:47-49.
Liu Z, Zhao B, Sun L, Wan X, Huo X. Research advances in swainsonine resources and its detection methods. Chinese Vet. Sci. 2008;38:175-180. doi: org/10.3969/j.issn.1673-4696.2008.02.019.
Bennett RB, Choi JR, Montgomery WD, Cha JK. A short, enantioselective synthesis of (−)-swainsonine. J. Am. Chem. Soc. 1989;111:2580-2582. doi: org/10.1021/ja00189a034.
Tong DW, Cao GR, Li SJ. Isolation and identification of swainsonine from Oxytropis kansuensis Bunge. J. Northwest A&F Univ. (Na.t Sci. Edit.) 2001;29:295-298.
Shi YH. Functional analysis of Metarhizium acridum MabrlA in conidiation regulation and pathogenicity. M. D. of Science, dissertation, Chongqing University, Chongqing, China, 2016.
Proctor RH, Hohn TM, McCormick SP. Reduced virulence of Gibberella zeae caused by disruption of a trichothecene toxin biosynthetic gene. Mol. Plant-Microbe Interact. 1995;8:593-601. doi: 10.1094/MPMI-8-0593.
Song R, Wang J, Sun L, Zhang Y, Ren Z, Zhao B, Lu H. The study of metabolites from fermentation culture of Alternaria oxytropis. BMC Microbiol. 2019;11:35. doi: org/10.1186/s12866-019-1408-8.

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The role of swnR gene on the biosynthesis pathway of the swainsonine in Metarhizium anisopliae

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