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Abstract Fusarium wilt of banana， caused by Fusarium oxysporum f. sp. cubense tropic race 4 （Foc TR4）， is a typical vascular and soilborne disease which has significantly threatened the sustainable development of banana industry. In order to reveal the infection process and pathogenesis of Foc TR4， the young mycelia （66.7 mg/ml） of wildtype strain of Foc TR4 （WTFoc TR4） cultured for 18-20 h were lysed with enzyme mixture for protoplast formation， which consisted of 25 mg/mldriselase， 0.4 mg/ml chitinase， 15 mg/ml lysing enzyme and 1.2 mol/L potassium chloride. The resulted protoplasts of 2×107 cells/ml were used to test the efficiency of transformation mediated by polyethylene glycol， and up to 9 transformants per microgram of DNA were obtained. AmCyan， RFP and YFP genes were stably transferred into the WTFoc TR4， separately， using the protoplast transformation system. The gene FoOCH1 encoding α1， 6mannosyltransferase in the WTFoc TR4 was knocked out using the splitmarker recombination technology. The genetic transformation and gene knockout system in this pathogen lays a foundation for the study of functional genomics and plantpathogen interactions.
Key words Fusarium wilt of banana; Fusarium oxysporum f. sp. cubense; Protoplasts; Transformation; Splitmarker recombination
Fusarium wilt of banana is a typical vascular bundle and soilborne fungal disease， as well as one of the most devastating plant diseases[1]. Once a banana plantlet is infected， it is difficult to fruit， and there are no effective prevention measures[1]. Therefore， to study the infection process and pathogenic mechanism of this pathogen can broaden our thinking to seek new ways to prevent and control this disease.
Fusarium wilt of banana is caused by Fusarium oxysporum f. sp. cubense （Foc）[1]. The Foc that has the greatest impact on production is the tropical race 4 （TR4） that infects almost all banana cultivars including the Gros Michel and the Cavendish， etc.[1].
At present， the genome sequencing work of more than 100 fungi has been completed， and the data was published[2]. Although the whole genome sequence of Foc race 4 has been determined[3]， only a few pathogenic genes such as FoOCH1[4]， FoSlt2， FoMkk2 and FoBck1[5]have been reported， and the pathogenesis of Fusarium wilt of banana is far from clear. Therefore， in this study， Foc TR4 was used and the protoplast preparation， transformation and gene knockout system were established， laying a foundation for the functional verification of F. oxysporum and plantpathogen interactions. Materials and Methods
The strain and plasmids
The Foc TR4 strain （151） with strong pathogenicity isolated from Xishuangbanna， Yunnan and the pZD 101AmCyan， pZD 101RFP and pZD 101YFP plasmids were used in this study.
Media and reagents
PDA medium： potato 200 g， glucose 20 g， agar powder 20 g and water 1 L; PDB medium： potato 200 g， glucose 20 g and water 1 L; YEPD medium： yeast extract 3 g， peptone 10 g， glucose 20 g and water 1 L; TB3/lowmeltingpoint TB3 medium： yeast extract 3 g， casein hydrolysate 3 g， sucrose 200 g， agarose/lowmeltingpoint agarose 7 g and water 1 L; STC buffer solution： 20% sucrose， 10 mmol/L TrisHCl， 50 mmol/L calcium chloride， pH 8.0; PTC buffer： 40% PEG4000 dissolved in STC buffer. Reagents： driselase （Sigma D9515）， chitinase （Sigma C6137）， lysing enzyme （Sigma L1412）， ampicillin （Sigma A9518） and hygromycin B （Sigma H3274）.
Enzyme mixture
25 mg/ml driselase， 0.4 mg/ml chitinase and 15 mg/ml lysing enzyme were dissolved in 1.2 mol/L potassium chloride solution and gently shaken at 120 r/min for 1 h， obtaining a mixture which was centrifuged at 8 000 r/min for 8 min. The supernatant was taken as an enzyme mixture for enzymolysis of mycelia.
Protoplast preparation
The protoplasts were produced according to the preparation method of F. graminearum protoplasts[6]with appropriate modifications. The conidia on the PDA plate cultured for 7 d were collected and injected into 100 ml of PDB medium， followed by culture at 150 r/min for 3 d at 25 ℃. The mycelia were removed by gauze filtration， and the liquid was centrifuged at 8 000 r/min for 10 min. The spores were collected and added into 100 ml of YEPD medium， and cultured at 175 r/min and 25 ℃ for 18-20 h; and after gauze filtration， 2 g of wet mycelia were measured and added into 30 ml enzyme mixture， followed by enzymolysis at 90 r/min for 1-3 h at 30 ℃. The protoplast formation was observed under a microscope every 0.5 h. When the protoplasts were produced in large quantities， the enzymatic hydrolysate was filtered with a nylon membrane having a pore size of 30 μm， and the liquid was centrifuged at 3 000 r/min for 5 min to collect the protoplast precipitate. After gently dissolving with 1.2 mol/L potassium chloride solution， centrifugation was performed at 3 000 r/min for 5 min， and the protoplasts were then dissolved by shaking in STC buffer. The concentration was then adjusted by the blood cell counting plate to 1×107 to 3×107 cells/ml. The protoplast liquid was filled into tubes according to 200 μl per tube and slowly frozen at -70 ℃. The assessment method of protoplast yield was to obtain the number of protoplasts per milliliter of enzymatic hydrolysate. Protoplast transformation
Protoplast transformation was performed according to the method of F. graminearum[6]with minor modifications. At first， 30 μl exogenous DNA fragment （about 1 μg） was added into 200 μlprotoplast， and the mixture was gently mixed and transferred to 10 ml EP tube， followed by standing for 15 min at room temperature. Then， 100， 200 and 700 μl of PTC buffer were added sequentially， followed by mixing gently and standing at room temperature for 15 min. Next， 5 ml of lowmeltingpoint TB3 medium （about 37 ℃， liquid form） was added， and the mixture was slightly inverted 3 times， and added into TB3 plates containing 100 μg/L ampicillin and 400 μg/ml hygromycin B. Culture was performed at 25 ℃ for 3 d. After further screening and purification of single colonies， two parts were picked from each colony. Specifically， one part was transferred to a PDA plate and cultured at 25 ℃ for 7 d， and the mycelial DNA was extracted for PCR detection; and the other was transferred to a PDA slant and cultured at 25 ℃ for 7 d， and then stored at 4 ℃. The evaluation method of protoplast transformation efficiency was to obtain the number of transformants obtained per microgram of DNA.
Gene knockout and transformant identification
Homologous recombination knockout was performed on FoOCH1[4]using the splitmarker recombination technology[7]（Fig. 1）. The primer names and sequences used are shown in Table 1.
Results and Analysis
Protoplast formation and transformation
2 g of young mycelia which were shaken for 18-20 h were added into 30 ml enzyme liquid for cultivating 2 h， and the protoplast yield wasabout 2×107 cells/ml. 30 μl plasmid DNA fragment （about 1 μg）digested with EcoR I was added into 200 μl of protoplasts （1×107 cells/ml） for transformation. After screening with 400 μg/ml hygromycin B， 14 transformants were obtained averagely， of which 9 transformants had bright fluorescence， and 5 transformants showed extremely weak fluorescence. The foreign DNA fragments constructed by splitmarker recombination were transformed in the same way， and 20 transformants were obtained on average.
It was verified that 9 knockout mutants and 11 false positive transformants were obtained. Fluorescence microscopy observation revealed that the autofluorescence of the wildtype mycelia was extremely weak （Fig. 2-A， Fig. 2-B）， while the pZD 101AmCyan transformantsemitted bright green fluorescence （Fig. 2-C，Fig. 2-D）， the pZD 101RFP transformants emitted red fluorescence （Fig. 2-E， Fig. 2-F）， and the pZD 101YFP transformants emitted yellow fluorescence （Fig. 2-G， Fig. 2-H）. A： Verification of AmCyanFoc TR4; M： DL2000 DNA Marker; 1-4： amplified with primers HyF/YgR; 5-8： amplified with primers AmCyanF/AmCyanR; 1， 5： plasmid pZD 101AmCyan; 2， 6： AmCyanFoc TR4; 3， 7： WTFoc TR4; 4，8： ddH2O. B： Verification of △FoOCH1; M： DL2000 DNA Marker; 1-4： amplified with primers InF/InR; 5-8： amplified with primers OutF/OutR; 1，2，5，6： △FoOCH1; 3， 7： WTFoc TR4; 4， 8： ddH2O.
Transformant PCR verification
The mycelial DNA of transformants with green fluorescence was extracted and amplified by primers HyF/YgR and AmCyanF/AmCyanR， respectively， and Hygromycin B and AmCyan gene fragments were obtained， respectively， indicating that the fluorescent genes were inserted into the genome of F. oxysporum （Fig. 3-A）. Primers InF/InR and OutF/OutR were used to identify the mycelial DNA of the knockout mutant. The amplification with internal primers gave no amplification product， and that with the outer primers produced the Hygromycin B gene fragments， demonstrating that FoOCH1 had been successfully knocked out by Hygromycin B （Fig. 3-B）.
Lei ZHANG et al. Construction of PEGmediated Genetic Transformation and Gene Knockout System in Fusarium oxysporum f. sp. cubense Tropic Race 4
Discussion
The protoplast yield in this study was approximately 2×107 cells/ml， and an average of 9 transformants were obtained per microgram of DNA. The values were 28% higher than the protoplast yield and transformation efficiency reported previously， respectively[8]. The protoplast transformation method of F. oxysporum was similar to that of F. graminearum. By this method， we obtained AmCyan， RFP and YFP fluorescent proteinlabeled F. oxysporum transformants， which proved that the system is universally applicable to filamentous fungi with sporulation. A knockout mutant of the FoOCH1 gene was obtained by protoplast transformation using the splitmarker recombination technology. If the protoplasts are further prepared by this mutant strain and transformed for gene knockout， a double or triple knockout mutant can be obtained by neomycin or nourseothricin resistance screening， and this system provides a basis for the study of fungal multigene synergy.
References
[1] PLOETZ RC. Management of Fusarium wilt of banana： A review with special reference to tropical race 4[J]. Crop Protection， 2015（73）： 7-15.
[2] SHARMA KK. Fungal genome sequencing： basic biology to biotechnology[J]. Critical Reviews in Biotechnology， 2016， 36（4）： 743-759. [3] GUO L， HAN L， YANG L， et al. Genome and transcriptome analysis of the fungal pathogen Fusarium oxysporum f. sp. cubense causing banana vascular wilt disease[J]. PLoS One， 2014， 9（4）： e95543.
[4] LI MH， XIE XL， LIN XF， et al. Functional characterization of the gene FoOCH1 encoding a putative alpha1， 6mannosyltransferase in Fusarium oxysporum f. sp. cubense[J]. Fungal Genetics & Biology， 2014（65）： 1-13.
[5] DING Z， LI M， SUN F， et al. Mitogenactivated protein kinases are associated with the regulation of physiological traits and virulence in Fusarium oxysporum f. sp. cubense[J]. PLoS One， 2015， 10（4）： e0122634.
[6] YUAN TL， ZHANG Y， YU XJ， et al. Optimization of transformation system of Fusarium graminearum[J]. Plant Physiology Communications， 2008， 44（2）： 251-256. （in Chinese）
[7] CATLETT NL， LEE BN， YODER OC， et al. Splitmarker recombination for efficient targeted deletion of fungal genes[J]. Fungal Genetics Reports， 2003， 50（1）： 9-11.
[8] XIE D， YANG Y， ZHOU D， et al. Red fluorescent protein gene （DsRed） transformation of Fusarium oxysporum f. sp. cubense race 4[J]. Chinese Journal of Tropical Crops， 2012， 33（4）： 685-689. （in Chinese）
Key words Fusarium wilt of banana; Fusarium oxysporum f. sp. cubense; Protoplasts; Transformation; Splitmarker recombination
Fusarium wilt of banana is a typical vascular bundle and soilborne fungal disease， as well as one of the most devastating plant diseases[1]. Once a banana plantlet is infected， it is difficult to fruit， and there are no effective prevention measures[1]. Therefore， to study the infection process and pathogenic mechanism of this pathogen can broaden our thinking to seek new ways to prevent and control this disease.
Fusarium wilt of banana is caused by Fusarium oxysporum f. sp. cubense （Foc）[1]. The Foc that has the greatest impact on production is the tropical race 4 （TR4） that infects almost all banana cultivars including the Gros Michel and the Cavendish， etc.[1].
At present， the genome sequencing work of more than 100 fungi has been completed， and the data was published[2]. Although the whole genome sequence of Foc race 4 has been determined[3]， only a few pathogenic genes such as FoOCH1[4]， FoSlt2， FoMkk2 and FoBck1[5]have been reported， and the pathogenesis of Fusarium wilt of banana is far from clear. Therefore， in this study， Foc TR4 was used and the protoplast preparation， transformation and gene knockout system were established， laying a foundation for the functional verification of F. oxysporum and plantpathogen interactions. Materials and Methods
The strain and plasmids
The Foc TR4 strain （151） with strong pathogenicity isolated from Xishuangbanna， Yunnan and the pZD 101AmCyan， pZD 101RFP and pZD 101YFP plasmids were used in this study.
Media and reagents
PDA medium： potato 200 g， glucose 20 g， agar powder 20 g and water 1 L; PDB medium： potato 200 g， glucose 20 g and water 1 L; YEPD medium： yeast extract 3 g， peptone 10 g， glucose 20 g and water 1 L; TB3/lowmeltingpoint TB3 medium： yeast extract 3 g， casein hydrolysate 3 g， sucrose 200 g， agarose/lowmeltingpoint agarose 7 g and water 1 L; STC buffer solution： 20% sucrose， 10 mmol/L TrisHCl， 50 mmol/L calcium chloride， pH 8.0; PTC buffer： 40% PEG4000 dissolved in STC buffer. Reagents： driselase （Sigma D9515）， chitinase （Sigma C6137）， lysing enzyme （Sigma L1412）， ampicillin （Sigma A9518） and hygromycin B （Sigma H3274）.
Enzyme mixture
25 mg/ml driselase， 0.4 mg/ml chitinase and 15 mg/ml lysing enzyme were dissolved in 1.2 mol/L potassium chloride solution and gently shaken at 120 r/min for 1 h， obtaining a mixture which was centrifuged at 8 000 r/min for 8 min. The supernatant was taken as an enzyme mixture for enzymolysis of mycelia.
Protoplast preparation
The protoplasts were produced according to the preparation method of F. graminearum protoplasts[6]with appropriate modifications. The conidia on the PDA plate cultured for 7 d were collected and injected into 100 ml of PDB medium， followed by culture at 150 r/min for 3 d at 25 ℃. The mycelia were removed by gauze filtration， and the liquid was centrifuged at 8 000 r/min for 10 min. The spores were collected and added into 100 ml of YEPD medium， and cultured at 175 r/min and 25 ℃ for 18-20 h; and after gauze filtration， 2 g of wet mycelia were measured and added into 30 ml enzyme mixture， followed by enzymolysis at 90 r/min for 1-3 h at 30 ℃. The protoplast formation was observed under a microscope every 0.5 h. When the protoplasts were produced in large quantities， the enzymatic hydrolysate was filtered with a nylon membrane having a pore size of 30 μm， and the liquid was centrifuged at 3 000 r/min for 5 min to collect the protoplast precipitate. After gently dissolving with 1.2 mol/L potassium chloride solution， centrifugation was performed at 3 000 r/min for 5 min， and the protoplasts were then dissolved by shaking in STC buffer. The concentration was then adjusted by the blood cell counting plate to 1×107 to 3×107 cells/ml. The protoplast liquid was filled into tubes according to 200 μl per tube and slowly frozen at -70 ℃. The assessment method of protoplast yield was to obtain the number of protoplasts per milliliter of enzymatic hydrolysate. Protoplast transformation
Protoplast transformation was performed according to the method of F. graminearum[6]with minor modifications. At first， 30 μl exogenous DNA fragment （about 1 μg） was added into 200 μlprotoplast， and the mixture was gently mixed and transferred to 10 ml EP tube， followed by standing for 15 min at room temperature. Then， 100， 200 and 700 μl of PTC buffer were added sequentially， followed by mixing gently and standing at room temperature for 15 min. Next， 5 ml of lowmeltingpoint TB3 medium （about 37 ℃， liquid form） was added， and the mixture was slightly inverted 3 times， and added into TB3 plates containing 100 μg/L ampicillin and 400 μg/ml hygromycin B. Culture was performed at 25 ℃ for 3 d. After further screening and purification of single colonies， two parts were picked from each colony. Specifically， one part was transferred to a PDA plate and cultured at 25 ℃ for 7 d， and the mycelial DNA was extracted for PCR detection; and the other was transferred to a PDA slant and cultured at 25 ℃ for 7 d， and then stored at 4 ℃. The evaluation method of protoplast transformation efficiency was to obtain the number of transformants obtained per microgram of DNA.
Gene knockout and transformant identification
Homologous recombination knockout was performed on FoOCH1[4]using the splitmarker recombination technology[7]（Fig. 1）. The primer names and sequences used are shown in Table 1.
Results and Analysis
Protoplast formation and transformation
2 g of young mycelia which were shaken for 18-20 h were added into 30 ml enzyme liquid for cultivating 2 h， and the protoplast yield wasabout 2×107 cells/ml. 30 μl plasmid DNA fragment （about 1 μg）digested with EcoR I was added into 200 μl of protoplasts （1×107 cells/ml） for transformation. After screening with 400 μg/ml hygromycin B， 14 transformants were obtained averagely， of which 9 transformants had bright fluorescence， and 5 transformants showed extremely weak fluorescence. The foreign DNA fragments constructed by splitmarker recombination were transformed in the same way， and 20 transformants were obtained on average.
It was verified that 9 knockout mutants and 11 false positive transformants were obtained. Fluorescence microscopy observation revealed that the autofluorescence of the wildtype mycelia was extremely weak （Fig. 2-A， Fig. 2-B）， while the pZD 101AmCyan transformantsemitted bright green fluorescence （Fig. 2-C，Fig. 2-D）， the pZD 101RFP transformants emitted red fluorescence （Fig. 2-E， Fig. 2-F）， and the pZD 101YFP transformants emitted yellow fluorescence （Fig. 2-G， Fig. 2-H）. A： Verification of AmCyanFoc TR4; M： DL2000 DNA Marker; 1-4： amplified with primers HyF/YgR; 5-8： amplified with primers AmCyanF/AmCyanR; 1， 5： plasmid pZD 101AmCyan; 2， 6： AmCyanFoc TR4; 3， 7： WTFoc TR4; 4，8： ddH2O. B： Verification of △FoOCH1; M： DL2000 DNA Marker; 1-4： amplified with primers InF/InR; 5-8： amplified with primers OutF/OutR; 1，2，5，6： △FoOCH1; 3， 7： WTFoc TR4; 4， 8： ddH2O.
Transformant PCR verification
The mycelial DNA of transformants with green fluorescence was extracted and amplified by primers HyF/YgR and AmCyanF/AmCyanR， respectively， and Hygromycin B and AmCyan gene fragments were obtained， respectively， indicating that the fluorescent genes were inserted into the genome of F. oxysporum （Fig. 3-A）. Primers InF/InR and OutF/OutR were used to identify the mycelial DNA of the knockout mutant. The amplification with internal primers gave no amplification product， and that with the outer primers produced the Hygromycin B gene fragments， demonstrating that FoOCH1 had been successfully knocked out by Hygromycin B （Fig. 3-B）.
Lei ZHANG et al. Construction of PEGmediated Genetic Transformation and Gene Knockout System in Fusarium oxysporum f. sp. cubense Tropic Race 4
Discussion
The protoplast yield in this study was approximately 2×107 cells/ml， and an average of 9 transformants were obtained per microgram of DNA. The values were 28% higher than the protoplast yield and transformation efficiency reported previously， respectively[8]. The protoplast transformation method of F. oxysporum was similar to that of F. graminearum. By this method， we obtained AmCyan， RFP and YFP fluorescent proteinlabeled F. oxysporum transformants， which proved that the system is universally applicable to filamentous fungi with sporulation. A knockout mutant of the FoOCH1 gene was obtained by protoplast transformation using the splitmarker recombination technology. If the protoplasts are further prepared by this mutant strain and transformed for gene knockout， a double or triple knockout mutant can be obtained by neomycin or nourseothricin resistance screening， and this system provides a basis for the study of fungal multigene synergy.
References
[1] PLOETZ RC. Management of Fusarium wilt of banana： A review with special reference to tropical race 4[J]. Crop Protection， 2015（73）： 7-15.
[2] SHARMA KK. Fungal genome sequencing： basic biology to biotechnology[J]. Critical Reviews in Biotechnology， 2016， 36（4）： 743-759. [3] GUO L， HAN L， YANG L， et al. Genome and transcriptome analysis of the fungal pathogen Fusarium oxysporum f. sp. cubense causing banana vascular wilt disease[J]. PLoS One， 2014， 9（4）： e95543.
[4] LI MH， XIE XL， LIN XF， et al. Functional characterization of the gene FoOCH1 encoding a putative alpha1， 6mannosyltransferase in Fusarium oxysporum f. sp. cubense[J]. Fungal Genetics & Biology， 2014（65）： 1-13.
[5] DING Z， LI M， SUN F， et al. Mitogenactivated protein kinases are associated with the regulation of physiological traits and virulence in Fusarium oxysporum f. sp. cubense[J]. PLoS One， 2015， 10（4）： e0122634.
[6] YUAN TL， ZHANG Y， YU XJ， et al. Optimization of transformation system of Fusarium graminearum[J]. Plant Physiology Communications， 2008， 44（2）： 251-256. （in Chinese）
[7] CATLETT NL， LEE BN， YODER OC， et al. Splitmarker recombination for efficient targeted deletion of fungal genes[J]. Fungal Genetics Reports， 2003， 50（1）： 9-11.
[8] XIE D， YANG Y， ZHOU D， et al. Red fluorescent protein gene （DsRed） transformation of Fusarium oxysporum f. sp. cubense race 4[J]. Chinese Journal of Tropical Crops， 2012， 33（4）： 685-689. （in Chinese）