Zinc finger transcription factor FoZfp1 is required for growth, conidiation, osmoregulation, and full virulence in the Polygonatum kingianum pathogen Fusarium oxysporum

Zinc finger transcription factor FoZfp1 is required for growth, conidiation, osmoregulation, and full virulence in the Polygonatum kingianum pathogen Fusarium oxysporum | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Zinc finger transcription factor FoZfp1 is required for growth, conidiation, osmoregulation, and full virulence in the Polygonatum kingianum pathogen Fusarium oxysporum Jianyun Su, Jingyi Wang, Jingying Tang, Weimei Yu, Jiajia Liu, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3899586/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 9 You are reading this latest preprint version Abstract Polygonatum kingianum rhizome rot is a destructive soil-borne disease caused by Fusarium oxysporum, which adversely affects the yield and sustainable development of P. kingianum. However, there are few effective control measures against rhizome rot. Thus, understanding the infection mechanism of F. oxysporum is essential to manage rhizome rot in P. kingianum effectively. In this study, zinc finger transcription factor FoZfp1 consisting of two C2H2 motifs was up-regulated during F. oxysporum conidial germination. The FoZfp1 gene deletion mutant (△FoZfp1) and the mutant complementary (△FoZfp1-C) strains were generated by the target gene replacement technique. Biological characteristic analyses revealed that the △FoZfp1 mycelial growth and conidial production were slower than those of the wild-type F. oxysporum (WT) and △FoZfp1-C. Additionally, the inhibition rates and sensitivity of △FoZfp1 under cell wall and osmotic targeted stresses were decreased compared to those of WT and △FoZfp1-C. Pathogenicity assays further revealed that the virulence of △FoZfp1 on the P. kingianum leaves and rhizomes was significantly reduced. These results indicate that FoZfp1 is associated with mycelial growth, conidiation, osmoregulation, and pathogenicity in F. oxysporum on P. kingianum. Biological sciences/Microbiology/Fungi/Fungal biology Biological sciences/Microbiology/Fungi/Fungal genetics Biological sciences/Microbiology/Fungi/Fungal pathogenesis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Polygonatum kingianum rhizome rot is a destructive soil-borne disease caused by Fusarium oxysporum, which adversely affects the yield and sustainable development of P. kingianum. However, there are few effective control measures against rhizome rot. Thus, understanding the infection mechanism of F. oxysporum is essential to manage rhizome rot in P. kingianum effectively. In this study, zinc finger transcription factor FoZfp1 consisting of two C2H2 motifs was up-regulated during F. oxysporum conidial germination. The FoZfp1 gene deletion mutant (△FoZfp1) and the mutant complementary (△FoZfp1-C) strains were generated by the target gene replacement technique. Biological characteristic analyses revealed that the △FoZfp1 mycelial growth and conidial production were slower than those of the wild-type F. oxysporum (WT) and △FoZfp1-C. Additionally, the inhibition rates and sensitivity of △FoZfp1 under cell wall and osmotic targeted stresses were decreased compared to those of WT and △FoZfp1-C. Pathogenicity assays further revealed that the virulence of △FoZfp1 on the P. kingianum leaves and rhizomes was significantly reduced. These results indicate that FoZfp1 is associated with mycelial growth, conidiation, osmoregulation, and pathogenicity in F. oxysporum on P. kingianum. Polygonatum kingianum Coll. et Hemsl. is a native medicinal plant in Yunnan province, whose rhizome has medicinal and dietary concomitant functions 1,2 . However, rhizome rot seriously threatens the sustainable production of P. kingianum . The causal agents of rhizome rot in P. kingianum are Fusarium oxysporum and F. solani, and the former is more virulent 3 . F. oxysporum is a destructive soil-borne vascular fungal pathogen and the fifth largest plant pathogenic fungus globally that causes fusarium wilt, root rot, and necrosis, leading to severe yield losses in over 100 host plants 4-6 . However, there are few effective control methods against F. oxysporum , given the long-term survival of its chlamydospores in the soil and mycelia colonization in host xylem vessels 7 . Therefore, understanding the pathogenic mechanism of rhizome rot in P. kingianum and screening and identifying the F. oxysporum pathogenic genes can provide clues for exploring the scientific prevention and control measures against rhizome rot. Transcription factors (TFs) regulate cell development, differentiation, and the cellular response to external perturbation by binding to a specific DNA site, or sites, where transcription activation or repression occurs through various mechanisms, including DNA–protein interactions, protein–protein interactions, and modification of the chromatin structure 8-10 . The zinc finger (ZF) protein is a TF with a ‘finger’ domain that regulates gene expression. It stabilizes a short polypeptide spatial configuration, folded into a finger-like structure by binding Zn 2+ . The ZF protein was first identified in Xenopus oocytes 11,12 , and is widely distributed in animals, plants and microorganisms 13 . It is divided into several subfamilies based on the numbers and positions of cysteine (Cys) and histidine (His) residues, including Cys 2 /His 2 -type (C 2 H 2 ), C 2 HC, C 2 C 2 , C 2 HCC 2 C 2 , and C 2 C 2 C 2 C 2 14 . More than 700 TFs have been predicted in F. oxysporum genome. However, only 26 TFs have been functionally analyzed, with the majority (15 TFs) belonging to the ZF protein family 15 . The 15 TFs include six Zn(II) 2 Cys 6 ZFs, five C 2 H 2 ZFs, two GATA ZFs, and two plant homeodomain (PHD)-containing ZFs 15 . The F. oxysporum homolog of the TF Ste12 possesses a C 2 H 2 domain, was up-regulated during the infection process, and was necessary for F. oxysporum virulence 16 . On the contrary, the pH signalling TF PacC with a C 2 H 2 domain negatively regulated virulence, preventing the transcription of acid-expressed genes essential during F. oxysporum infection 17 . Zinc homeostasis regulator ZafA is also a C 2 H 2 ZF. It was significantly up-regulated during the early stages of infection and was required for the full virulence of F. oxysporum, especially when zinc was limited 18 . However, FolCzf1 , a C 2 H 2 ZF in F. oxysporum f. sp. lycopersici ( Fol ), was required for growth, conidiation, conidia morphology, and pathogenicity in tomato 19 . Similarly, the Con7-1 (a C 2 H 2 ZF in F. oxysporum ) deletion mutant exhibited defects in chitin synthase, hyphal branch, conidiation, and virulence 20 . Although these five C 2 H 2 ZFs have been identified and functionally tested, this is not even close to sufficient. Therefore, this study aimed to investigate the roles this new ZF protein TF FoZfp1 with two C 2 H 2 domains in the developmental processes and pathogenicity of the F. oxysporum in P. kingianum . The FoZfp1 deletion mutants ( △ FoZfp1 ) and the mutant complementary ( △ FoZfp1 -C) strains were generated by the target gene replacement technique. The FoZfp1 was up-regulated during F. oxysporum conidial germination . The inhibition rates, sensitivity under cell wall and osmotic targeted stresses, and virulence of △ FoZfp1 were decreased compared to those of WT and △ FoZfp1 -C. The findings from this study elaborate on the effects of FoZfp1 on F. oxysporum growth, conidiation, response against stress, and virulence . Results Assessment of FoZfp1 expression pattern during conidial germination Transcriptome analysis revealed that the FoZfp1 expression was up-regulated in F. oxysporum conidia cultured for 12 h and 24 h compared to the control (0 h) 21 . The qRT-PCR results revealed that FoZfp1 was up-regulated during F. oxysporum conidial germination (Fig. 1). FoZfp1 is a C 2 H 2 -type ZF protein BLASTn analysis revealed that FoZfp1 nucleotide sequence (OR715798) was 99% identical to F. oxysporum f. sp. lycopersici 4287 ZF protein MSN2/4 (XM_018379112) 22 . Additionally, FoZfp1 was classified into the C 2 H 2 -type subfamily, Ste12 (ACM80357) 16 , containing two C 2 H 2 zinc finger domains (Fig. 2A, B), making it a C 2 H 2 -type ZF protein. Verification of FoZfp1 deletion and complementary mutants The hygromycin B-resistant transformants were verified by PCR. The FoZfp1 gene was successfully replaced in the FoZfp1 transformants ( △ FoZfp1 ) (Fig. 3A). Co-transformation of the FoZfp1 gene with the vector pDHtsk-GFP-G418 successfully complemented the FoZfp1 gene ( △ FoZfp1 -C). Additionally, the target FoZfp1 nucleotide sequences were amplified in WT and △ FoZfp1 -C but not in △ FoZfp1 (Fig. 3B) . At the same time, the hygromycin B-resistance gene were amplified in △ FoZfp1 and △ FoZfp1 -C but not in WT (Fig. 3C). Furthermore, the neomycin-resistance gene were amplified in △ FoZfp1 -C but not in WT and △ FoZfp1 (Fig. 3D). Validation of the FoZfp1 expression levels by qRT-PCR revealed that △ FoZfp1 expression was significantly reduced compared to WT and △ FoZfp1 -C expressions (Fig. 3E) . These results indicated that FoZfp1 was knocked out in △ FoZfp1 and was complemented in △ FoZfp1 -C. Deletion of FoZfp1 affects mycelial growth and conidial formation The △ FoZfp1 colony formation and growth rates were significantly slow and reduced compared to WT and △ FoZfp1 -C cultured on potato dextrose agar (PDA) for 5 days (Fig. 4A, B). Furthermore, a significantly reduced number of micro-conidia was produced in △ FoZfp1 at 96 h post-inoculation on potato dextrose broth (PDB) (Fig. 4C). However, △ FoZfp1 spore morphology was not different compared to WT and △ FoZfp1 -C (Fig. 4D). These results indicated that deletion of FoZfp1 affected mycelial growth and conidial formation in F. oxysporum . Deletion of FoZfp1 impairs its pathogenicity In the infection assays, the △ FoZfp1 lesion diameters in the detached leaves and rhizomes were significantly decreased compared to WT and △ FoZfp1 -C (Fig. 5A, B). Withering of the leaves and rhizome rot were observed among the P. kingianum plants inoculated with WT and △ FoZfp1 -C. However, no symptoms were observed in △ FoZfp1 (Fig. 5C-E). Overall, the disease index of WT and △ FoZfp1 -C was significantly higher than that of △ FoZfp1 (Fig. 5F) . These results demonstrated that FoZfp1 was required for full virulence in F. oxysporum on P. kingianum. FoZfp1 contributes to F. oxysporum stress responses The inhibition rates and sensitivity of △ FoZfp1 under cell wall (sodium dodecyl sulfate, SDS; congo red, CR), osmotic stress (NaCl, KCl), and tebuconazole stresses were decreased compared to WT and △ FoZfp1 -C. However, △ FoZfp1 had a lower tolerance to carbendazim compared to WT and △ FoZfp1 -C. Besides, the differences in the inhibition rates on △ FoZfp1, WT, and △ FoZfp1 -C were insignificant under oxidative stress (H 2 O 2 ), suggesting that FoZfp1 had little effect on the sensitivity to oxidative stress (Fig. 6). These results suggested that FoZfp1 was involved in regulating responses to osmotic pressure and cell wall integrity stresses in F. oxysporum . Discussion Rhizome rot is a devastating soil-borne disease, seriously threatening the P. kingianum industry. F. oxysporum invades the roots, causing wounds, colonizes the vascular tissues, blocking water and nutrient transport , and may even lead to plant death 23 . Although over 700 TFs have been predicted in F. oxysporum , only 15 ZFs are associated with pathogenicity 15 . Among them is Fow2 , a Zn(II) 2 Cys 6 -type transcription regulator, essential for root invasion and colonization but not for vegetative growth and conidiation in F. oxysporum 24 . The cutinase transcription factors ctf1 and ctf2 , containing the Zn 2 Cys 6 DNA binding domain, also play important roles in the lipolytic system of Fol . Additionally, ctf1 and ctf2 deletion mutants severely reduce F. oxysporum virulence 25 . In common bean, Fusarium transcription factor 1 ( ftf1 ) with a Zn(II) 2 Cys 6 motif is only up-regulated during plant infection, where multiple ftf1 copies increase virulence in F. oxysporum f. sp. phaseol 26 . Additionally, EBR1 , belonging to the Zn 2 Cys 6 family, regulates the expression of genes encoding metabolism and virulence. EBR1 deletion impairs growth and reduces pathogenicity and biocontrol capacities in different F. oxysporum strains 27 . Furthermore, the global nitrogen regulator FNR1 , with a single conserved GATA-type ZF domain, regulates the secondary nitrogen acquisition in plants. Notably, the disruption of FNR1 mutants significantly delays F. oxysporum infection in tomato seedlings 28 . Besides, Cti6 , which contains a PHD finger motif and simultaneously interacts with the transcriptional corepressor complex Cyc8-Tup1 and the co-activator SAGA (Spt-Ada-Gcn5-acetytransferase) complex, is required for full virulence in F. oxysporum on tomato 29 . In this study, C 2 H 2 ZF FoZfp1 was identified, and it was required for full virulence in F. oxysporum on P. kingianum . However, the regulatory mechanism in pathogenicity needs to be analyzed in the plant-pathogen interaction. The host immune system first detects the pathogenic fungi conidia during the infection process. With F. oxysporum , conidial germination is the key step of infection 30 . Therefore, early conidial detection is crucial to inhibit fungal growth and alleviate the disease occurrence 31 . In this study, C 2 H 2 ZF FoZfp1 was screened using the genome and transcriptome data of the conidial germination process in F. oxysporum . FoZfp1 was significantly up-regulated during conidial germination, suggesting that this gene might be related to F. oxysporum growth and pathogenicity. To verify this conjecture, FoZfp1 and the mutant complementary strains were constructed using split-marker homologous recombination, which revealed that FoZfp1 regulated mycelial growth and conidial yield. However, this did not affect the conidia morphology. Similarly, BcTaf14 , TATA box-binding protein-associated factor 14 (Taf14) in Botrytis cinerea was associated with mycelial growth and conidial morphogenesis with no effect on conidial germination ratio and rate 32 . snt2 , a PHD-containing ZF, was also essential in vegetative growth, conidial production, and host colonization by F. oxysporum f. sp. melonis 33 . However, the white-collar 1 photoreceptor Wc1 , a GATA ZF, and knockout mutants of F. oxysporum lacking Wc1 impaired aerial hyphae and virulence 34 . When a pathogen invades the host, the host exhibits some defensive response, including a change in the internal environment or vascular system 35 . Similarly, pathogenic fungi always adopt a series of complex strategies for successful invasion of the host, including the plant defense mechanisms, host intracellular environment, and defeating adverse environmental changes 36,37 . In this study, △ FoZfp1 increased resistance to membrane stressor (SDS), cell wall stressor (CR), extracellular osmotic (NaCl), and intracellular osmotic (KCl) stress compared to the WT strain. However, no differences were identified in tolerance to oxidative (H 2 O 2 ) stress between the mutants and the WT strains, implying that FoZfp1 regulated osmotic pressure and cell wall integrity stresses. These results are similar to those of Aoime2 ( Arthrobotrys oligospora inducer of meiosis 2) deletion mutants, unaffected by oxidative stressor H 2 O 2 but highly sensitive to the osmotic stressor NaCl 38 . Similarly, BcTaf14 deletion mutants increased NaCl and KCl sensitivity 32 . Neutral trehalase-encoding gene NTH1 knockout mutant was also sensitive to H 2 O 2 and SDS but not to CR, NaCl, and KCl 39 . In conclusion, the C 2 H 2 ZF-encoding gene FoZfp1 plays significant roles in mycelial growth, conidiation, stress response, and pathogenicity in F. oxysporum of P. kingianum. However, how FoZfp1 exerts these functions requires further study. Materials And Methods Isolation of the fungal strain and the culture conditions The wild-type F. oxysporum strain PkF01 isolated from the P. kingianum rhizome rot samples, was identified with nucleotide sequences of the elongation factor 1-alpha (MW149127) and the second largest subunit of nuclear DNA-directed RNA polymerase II (MW194100) by L. Zhang in our previous study 3 . For conidia production, mycelia were incubated in PDB at 28°C with shaking at 180 revolutions per minute (rpm) for 3 days. Subsequently, the conidial suspension was adjusted to 1×10 6 conidia·mL -1 , and 30% glycerol was added before storing the suspension at −80°C 40 . Phylogenetic tree construction and protein sequence alignment FoZfp1 nucleotide sequence was submitted to the National Center for Biotechnology Information (NCBI) GenBank database, and BLASTn analysis was performed in NCBI. To further investigate the function of FoZfp1 in F. oxysporum , phylogenetic tree and alignment of the C 2 H 2 zinc-finger proteins of F. oxysporum were performed using the maximum likelihood method with 1,000 replications of bootstrap in MEGA 11 41 and edited in GeneDoc 42 , respectively. Generation of FoZfp1 deletion mutants ( △ FoZfp1 ) The FoZfp1 gene in WT was deleted using the split-marker recombination technology 43,44 . Firstly, 658 bp upstream ( FoZfp1 -Up) and 807 bp downstream ( FoZfp1 -Down) FoZfp1 fragments, and 800 bp upstream (Hy) and 1,112 bp downstream (Yg) hygromycin B-resistance cassette (HYG) fragments from the vector, pZD101-AmCyan 44 were amplified using four sets of primers FoZfp1 -UF/ FoZfp1 -UR, FoZfp1 -DF/ FoZfp1 -DR, Hy-F/Hy-R, and Yg-F/Yg-R, respectively. Secondly, FoZfp1 -Up and Hy were fused through PCR splicing by overlap extension 45 , using the FoZfp1 -UF/Hy-R primer pair and FoZfp1 -Up/Hy as the templates. Additionally, FoZfp1 -Down was fused with Yg using the Yg-F/ FoZfp1 -DR primer pair and FoZfp1 -Down/Yg as the template. Thirdly, the two fusion fragments were transformed into WT protoplasts following the polyethylene glycol-mediated protoplast transformation technique 46 . Finally, the transformants were screened on PDA containing ampicillin (100 mg·L -1 ) and hygromycin B (400 mg·L -1 ). Complementation of FoZfp1 deletion mutant ( △ FoZfp1-C ) △ FoZfp1 was complemented with a fragment containing FoZfp1 full-length cDNA. The fragment amplified with FoZfp1 -CF/ FoZfp1 - CR primer pair was cloned into the T3 promoter vector pDHtsk-GFP-G418 with a neomycin-resistant cassette. Next, the recombinant plasmid was transformed into △ FoZfp1 protoplasts. Subsequently, the transformants were screened on PDA containing hygromycin B (400 mg·L -1 ) and neomycin (300 mg·L -1 ). Verification and quantification of gene expression The deletion mutants were verified by PCR using primer pair FoZfp1 -IF/ FoZfp1 -IF, FoZfp1 -UH-F/ FoZfp1 -UH-R, and FoZfp1- DY-F /FoZfp1- DY-R. The complementary mutants were verified using FoZfp1 -IF/ FoZfp1 -IR, Hy -F /Yg -R, and Neo -F /Neo -R primer pairs. qRT-PCR further validated foZfp1 expression. RNA extraction, cDNA synthesis, and qRT-PCR were performed with TaKaRa MiniBEST Universal RNA Extraction Kit (TaKaRa, Code No. 9767), PrimeScript™ RT Master Mix (TaKaRa, Code No. RR036A), and TB Green® Premix Ex Taq™ II (TaKaRa, Code No. RR820A) according to manufacturer's instructions, respectively. The qRT-PCR conditions were as follows: initial denaturation at 95°C for 30s, followed by 40 cycles of 95°C for 5 s, and annealing at 60°C for 30 s. Elongation factor 1-alpha ( EF1α ) and tubulin 2 ( TUB2 ) were used as internal reference genes 47 . The relative expression of the target gene was calculated by the 2 – △△ Ct method 48 . The qRT-PCR assay was conducted with three independent biological replicates. The FoZfp1 gene expression were quantified by qRT-PCR using primer pair FoZfp1 -QF1/ FoZfp1 -QR1, EF1α -QF/ EF1α -QR, and TUB2 -QF/ TUB2 -QR. All primers used in this study were listed in Table S1. Mycelial growth and conidiation assays For mycelial growth, a 5-mm-diameter mycelial plug from a 3-day-old culture was placed on PDA and incubated at 28°C for 5 days. For 5 days, the colony morphology was photographed, and the colony diameter was measured daily. For conidiation, a 5-mm-diameter mycelial plug from a 3-day-old culture was placed on PDB (200 mL) with shaking at 180 rpm and 28°C. The conidial yield was calculated at 48, 72, and 96 h post-inoculation on PDB using a blood counting chamber. Subsequently, conidia were filtered through two layers of lens paper and resuspended to a concentration of 1×10 6 spores·mL -1 in PDB. The conidia germination rate was calculated at 12 h post resuspension. The assays were performed with three biological and three technical replicates. Pathogenicity assays P. Kingianum plants collected from plantation in Kunming city of Yunnan province, China, and were permitted and identified by P. Ji from Institute of Medicinal Plant Cultivation. One drop of conidial suspension (1×10 6 spores·mL -1 ) was dripped onto the surface of each P. kingianum leaf and tuber 49,50 . Leaves/rhizomes inoculated with sterile water were used as the controls. Inoculated leaves and rhizomes were cultured on moist filter paper at 28°C and 16-h light/8-h dark. Ten leaves and rhizomes were used for each treatment, with three biological replicates. Lesion diameters were measured at 5 days post-inoculation. Moreover, the roots of one-year-old P. kingianum plants were dipped into conidial suspension (1×10 6 spores·mL -1 ) for 30 min. Plants whose roots were dipped in sterile water were used as the control. Subsequently, the treatment and control plants were transplanted in pots filled with sterile soil and maintained in a growth chamber at 28°C, 60% relative humidity, and 16-h light/8-h dark for 30 d. Disease index was calculated using the formula: [∑(grade × number of plants corresponding grade) / (4 × total number of plants investigated )] × 100. Grade: 0 = healthy plants; 1 = yellowing of the lower leaves; 2 = yellowing of upper leaves; 3 = yellowing of most of the leaves; 4 = severe wilting or plant death 51 . Experimental studies on plant samples, including the supply of plant material, comply with institutional, national and international guidelines and legislation Response against stress A 5-mm-diameter mycelial plug from a 3-day-old culture was placed on PDA supplemented with 1.8 M NaCl, 1.8 M KCl, 0.05% SDS, 0.1% CR, 0.08% H 2 O 2 , 0.25 μg·mL -1 tebuconazole, and 0.4 μg·mL -1 carbendazim. All the cultures were incubated in the dark at 28°C. Subsequently, their colony diameter was measured after 5 days. The inhibition ratio (%) was calculated as (C-N)/C × 100 52 , where C is the colony diameter of control and N is the colony diameter of the treatment. All treatments and the control had three biological and technical replicates. Statistical analysis The analysis of variance (ANOVA) was performed by IBM SPSS Statistics 26 (IBM Corporation, USA). Significance in all the comparisons among means with standard deviation was calculated by ANOVA with Duncan’s multiple comparison adjustment. Diagrams were made by GraphPad Prism 8 (GraphPad Prism Software Inc., San Diego, CA). Declarations Authors contributions Conceptualization, L.Z; Data curation, J. S.; Formal analysis, J.W., J.T., J.L., X.D. and J.D.; Funding acquisition, L.Z.; Methodology, J.W., J.T. and W.Y.; Project administration, L.Z.; Writing – original draft, J.S.; Writing – review & editing, X.C., P.J. and L.Z. All authors have read and approved the manuscript. Funding This research was funded by National Natural Science Foundation of China (82360746), Yunnan Fundamental Research Projects (202101AT070245), Yunnan Provincial Science and Technology Department-Applied Basic Research Joint Special Funds of Yunnan University of Chinese Medicine (202101AZ070001-054), Academician (Expert) Workstation Project, Wang Yuan Chao Expert Workstation in Yunnan Province (202305AF150018), Yunnan Science and Technology Talent and Platform Program (202105AG070012), and Team Project of College of Chinese Materia Medica, Yunnan University of Chinese Medicine. Competing interests Te authors declare no competing interests. Data availability The datasets generated and/or analysed during the current study are available from the corresponding author on reasonable request. Additional information Supplementary Information Table S1 List of primers used in this study. Correspondence and requests for materials should be addressed to L.Z. References Zhao, P. et al. 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Plant Pathol. 22, 1239–1255. https://doi.org/https://doi.o rg/ 10.1111/mpp.13108 (2021). Zhang, L. et al. Identification and evaluation of resistance to Fusarium oxysporum f. sp. cubense tropical race 4 in Musa acuminata Pahang. Euphytica 214, 106. https://doi.org/10.1007/s10681-018-2185-4 (2018). Xiao, J. et al. Protein kinase Ime2 is associated with mycelial growth, conidiation, osmoregulation, and pathogenicity in Fusarium oxysporum . Arch. Microbiol. 204, 455. https://doi.org/10.1007/s00203-022-02964-0 (2022). Additional Declarations No competing interests reported. Supplementary Files TableS1ListofprimersusedinthisstudySR.docx Table S1 List of primers used in this study. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3899586","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":272829591,"identity":"ae715e01-7879-486d-9d26-6dd98e6b57ed","order_by":0,"name":"Jianyun Su","email":"","orcid":"","institution":"Academy of Southern Medicine, Yunnan University of Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Jianyun","middleName":"","lastName":"Su","suffix":""},{"id":272829592,"identity":"7672f190-2bef-4cdc-a4dc-ef2caedbed81","order_by":1,"name":"Jingyi Wang","email":"","orcid":"","institution":"Academy of Southern Medicine, Yunnan University of Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Jingyi","middleName":"","lastName":"Wang","suffix":""},{"id":272829593,"identity":"38d89a81-0ec2-402f-9a2b-7bad0e641281","order_by":2,"name":"Jingying Tang","email":"","orcid":"","institution":"Academy of Southern Medicine, Yunnan University of Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Jingying","middleName":"","lastName":"Tang","suffix":""},{"id":272829594,"identity":"d46160cd-322c-4bb6-aff2-633e8d106c39","order_by":3,"name":"Weimei Yu","email":"","orcid":"","institution":"Academy of Southern Medicine, Yunnan University of Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Weimei","middleName":"","lastName":"Yu","suffix":""},{"id":272829595,"identity":"e69336d0-9642-4a01-86be-a17ad8777a0a","order_by":4,"name":"Jiajia Liu","email":"","orcid":"","institution":"Academy of Southern Medicine, Yunnan University of Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Jiajia","middleName":"","lastName":"Liu","suffix":""},{"id":272829596,"identity":"bbf16cee-1b90-4fe7-a907-fdc1ea3264a6","order_by":5,"name":"Xian Dong","email":"","orcid":"","institution":"Academy of Southern Medicine, Yunnan University of Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Xian","middleName":"","lastName":"Dong","suffix":""},{"id":272829597,"identity":"614358f0-9775-4c79-a7d6-301554705948","order_by":6,"name":"Jiahong Dong","email":"","orcid":"","institution":"Academy of Southern Medicine, Yunnan University of Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Jiahong","middleName":"","lastName":"Dong","suffix":""},{"id":272829598,"identity":"b05e5ca5-caa8-41e9-9339-56c85c60e635","order_by":7,"name":"Xia Chai","email":"","orcid":"","institution":"Yunnan Normal University","correspondingAuthor":false,"prefix":"","firstName":"Xia","middleName":"","lastName":"Chai","suffix":""},{"id":272829599,"identity":"0b95f505-8327-4fd3-8904-2a8a6d63f453","order_by":8,"name":"Pengzhang Ji","email":"","orcid":"","institution":"Academy of Southern Medicine, Yunnan University of Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Pengzhang","middleName":"","lastName":"Ji","suffix":""},{"id":272829600,"identity":"ecbaede1-345c-452e-8a68-564565aa8922","order_by":9,"name":"Lei Zhang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAxElEQVRIie3QMQrCMBSA4VcCnaJdn0vrEQKF0qE3cUkQurk7FEkp2KXgKughCl7AEnD1CjmCbhmNo4Mkbg75hzfl4/ECEAr9Y0gkcINpQojSniSSIIYyX/RxzfwJxFsx3ukSvUR2alutKea5osCgqVZOEp2njvES00LNrhpu9Ua6CEGxR263FGrOWSSVm8QoesPtvHSUoRehdgu8yUh8CaLoUAyYo7KfzH1uyY7r6WnMLk0OSulHU7nJZ/y356FQKBT61gt5GDr4HbFITAAAAABJRU5ErkJggg==","orcid":"","institution":"Academy of Southern Medicine, Yunnan University of Chinese Medicine","correspondingAuthor":true,"prefix":"","firstName":"Lei","middleName":"","lastName":"Zhang","suffix":""}],"badges":[],"createdAt":"2024-01-26 09:44:15","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3899586/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3899586/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":51232159,"identity":"4a0e01dd-5e39-4d93-8cce-75a7c56c99d3","added_by":"auto","created_at":"2024-02-16 14:44:42","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":50668,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eFoZfp1\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e expression pattern at 0, 12, and 24 h of conidia germination. \u003c/strong\u003eError bars represent standard deviation of the mean with three independent biological replicates. TPM means trans per kilobase of exon model per million mapped reads.\u003c/p\u003e","description":"","filename":"Figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3899586/v1/7c7e40cac88fd7e344ca1f49.jpg"},{"id":51232162,"identity":"70165582-abf0-401f-8a78-63de5ce0b3c7","added_by":"auto","created_at":"2024-02-16 14:44:42","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":463926,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePhylogenetic relationships among \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eF. oxysporum\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e ZFs.\u003c/strong\u003e (A) Phylogenetic tree constructed based on amino acid sequences. Heat shock factor (HSF)-type TF was used as the outgroup. (B) Alignment of the C\u003csub\u003e2\u003c/sub\u003eH\u003csub\u003e2\u003c/sub\u003e ZF region of \u003cem\u003eF. oxysporum\u003c/em\u003e. Absolutely conserved residues were marked in black, and at least 80% of the residues were indicated in gray. Black lines indicate the two C\u003csub\u003e2\u003c/sub\u003eH\u003csub\u003e2\u003c/sub\u003e zinc-finger domains.\u003c/p\u003e","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3899586/v1/f4fc4dcea288a8ccdcb233df.jpg"},{"id":51232161,"identity":"4492e6b0-52ac-4499-a02a-924d7ab97d84","added_by":"auto","created_at":"2024-02-16 14:44:42","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":352428,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eVerification of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003e△FoZfp1\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eand \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003e△FoZfp1\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e-C mutants. \u003c/strong\u003e(A): Verification of \u003cem\u003e△FoZfp1\u003c/em\u003e-1 and \u003cem\u003e△FoZfp1\u003c/em\u003e-2 mutants by PCR.\u003cstrong\u003e \u003c/strong\u003e1: \u003cem\u003e△FoZfp1\u003c/em\u003eamplified using primer pair of \u003cem\u003eFoZfp1\u003c/em\u003e-IF/\u003cem\u003eFoZfp1\u003c/em\u003e-IR; 2: Hy-F/Yg-R; 3: \u003cem\u003eFoZfp1\u003c/em\u003e-UH-F\u003cem\u003e/FoZfp1\u003c/em\u003e-UH-R; 4: \u003cem\u003eFoZfp1\u003c/em\u003e-DY-F/\u003cem\u003eFoZfp1\u003c/em\u003e-DY-R. (B): \u003cem\u003eFoZfp1 \u003c/em\u003eamplification with \u003cem\u003eFoZfp1\u003c/em\u003e-IF/\u003cem\u003eFoZfp1\u003c/em\u003e-IR. (C): Hygromycin B-resistant\u003cem\u003e \u003c/em\u003egene amplification with Hy-F/Yg-R. (D): Neomycin-resistant genes\u003cem\u003e \u003c/em\u003eamplification with Neo-F/Neo-R. 1: \u003cem\u003e△FoZfp1\u003c/em\u003e-1;\u003cem\u003e \u003c/em\u003e2: \u003cem\u003e△FoZfp1\u003c/em\u003e-2; 3: \u003cem\u003e△FoZfp1\u003c/em\u003e-3. (E): Relative expression of \u003cem\u003eFoZfp1\u003c/em\u003e in WT, \u003cem\u003e△FoZfp1,\u003c/em\u003eand \u003cem\u003e△FoZfp1\u003c/em\u003e-C. Error bars represent standard deviation of the mean with three independent biological replicates. Different letters indicate significant differences at \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"Figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3899586/v1/8c8adfd0973cb20d2a90a7a9.jpg"},{"id":51232165,"identity":"03b357eb-aea3-4131-8f10-12283abd97ad","added_by":"auto","created_at":"2024-02-16 14:44:42","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":598755,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eWT, \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003e△FoZfp1\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e, and \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003e△FoZfp1\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e-C mycelial growth and conidiation.\u003c/strong\u003e (A): Colony morphology on PDA; (B): Colony diameters at 5 days post-inoculation on PDA; (C): Conidiation in PDB. (D): WT, \u003cem\u003e△FoZfp1\u003c/em\u003e, and \u003cem\u003e△FoZfp1\u003c/em\u003e-C conidial germination. Error bars represent standard deviation of the mean with three independent biological replicates. Different letters indicate significant differences at \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"Figure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3899586/v1/62d009f5e56dc31677899e71.jpg"},{"id":51232598,"identity":"5b904ea2-422a-4011-a789-90971b192210","added_by":"auto","created_at":"2024-02-16 14:52:42","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":857643,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eWT, \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003e△FoZfp1\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e, and \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003e△FoZfp1\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e-C virulence in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eF. oxysporum\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e. \u003c/strong\u003e(A): Symptoms on detached leaves and rhizomes inoculated with WT, \u003cem\u003e△FoZfp1\u003c/em\u003e, and \u003cem\u003e△FoZfp1\u003c/em\u003e-C for 5 days. (B): Lesion diameters of leaves and rhizomes inoculated with WT, \u003cem\u003e△FoZfp1\u003c/em\u003e, and \u003cem\u003e△FoZfp1\u003c/em\u003e-C after 5 days of inoculation. (C): Symptoms of plants inoculated with WT, \u003cem\u003e△FoZfp1\u003c/em\u003e, and \u003cem\u003e△FoZfp1\u003c/em\u003e-C after 15 days. (D): Symptoms of plants inoculated with WT, \u003cem\u003e△FoZfp1\u003c/em\u003e, and \u003cem\u003e△FoZfp1\u003c/em\u003e-C after 25 days. (E): Symptoms of plants inoculated with WT, \u003cem\u003e△FoZfp1\u003c/em\u003e, and \u003cem\u003e△FoZfp1\u003c/em\u003e-C after 30 days. (F): Disease index at 30 days post-inoculation. Scale bars: 10 mm. Error bars represent standard deviation of the mean with three independent biological replicates. Different letters indicate significant differences at \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"Figure5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3899586/v1/af3d3256287f0bf8905691d6.jpg"},{"id":51232163,"identity":"9255462f-ea1a-49f7-bdfa-4e19a61da3ca","added_by":"auto","created_at":"2024-02-16 14:44:42","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":665923,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eWT, \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003e△FoZfp1\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e, and \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003e△FoZfp1\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e-C vegetative growth under different stressors. \u003c/strong\u003e(A) WT, \u003cem\u003e△FoZfp1\u003c/em\u003e, and \u003cem\u003e△FoZfp1\u003c/em\u003e-C colony morphology on PDA (mock) and PDA supplemented with 0.1% CR, 0.08% H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, 0.05% SDS, 1.8 M KCl, 1.8 M NaCl, 0.4 μg·mL\u003csup\u003e-1\u003c/sup\u003e carbendazim, and 0.25 μg·mL\u003csup\u003e-1\u003c/sup\u003e tebuconazole for 5 days. (B) The relative inhibition rates based on colony diameters 5 days post-incubation. Error bars represent standard deviation of the mean with three independent biological replicates. Different letters indicate significant differences at \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"Figure6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3899586/v1/d8e0253ac8dea1e7b33d5c27.jpg"},{"id":51232742,"identity":"4f7faa9a-dcb4-44d8-8497-d7dd90aa5b76","added_by":"auto","created_at":"2024-02-16 15:00:42","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1200662,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3899586/v1/25133623-f854-4e09-b1d2-7d05b5d0a422.pdf"},{"id":51232597,"identity":"bc3e4a60-01ff-4a99-a600-6d6511736f21","added_by":"auto","created_at":"2024-02-16 14:52:42","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":14964,"visible":true,"origin":"","legend":"\u003cp\u003eTable S1 List of primers used in this study.\u003c/p\u003e","description":"","filename":"TableS1ListofprimersusedinthisstudySR.docx","url":"https://assets-eu.researchsquare.com/files/rs-3899586/v1/95e0de83f309bd76b8ade121.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Zinc finger transcription factor FoZfp1 is required for growth, conidiation, osmoregulation, and full virulence in the Polygonatum kingianum pathogen Fusarium oxysporum","fulltext":[{"header":"Introduction","content":"\u003cp\u003e\u003cspan style=\"text-align: start;color: rgb(25, 40, 55);background-color: rgb(255, 255, 255);font-size: 14.8px;\"\u003ePolygonatum kingianum rhizome rot is a destructive soil-borne disease caused by Fusarium oxysporum, which adversely affects the yield and sustainable development of P. kingianum. However, there are few effective control measures against rhizome rot. Thus, understanding the infection mechanism of F. oxysporum is essential to manage rhizome rot in P. kingianum effectively. In this study, zinc finger transcription factor FoZfp1 consisting of two C2H2 motifs was up-regulated during F. oxysporum conidial germination. The FoZfp1 gene deletion mutant (△FoZfp1) and the mutant complementary (△FoZfp1-C) strains were generated by the target gene replacement technique. Biological characteristic analyses revealed that the △FoZfp1 mycelial growth and conidial production were slower than those of the wild-type F. oxysporum (WT) and △FoZfp1-C. Additionally, the inhibition rates and sensitivity of △FoZfp1 under cell wall and osmotic targeted stresses were decreased compared to those of WT and △FoZfp1-C. Pathogenicity assays further revealed that the virulence of △FoZfp1 on the P. kingianum leaves and rhizomes was significantly reduced. These results indicate that FoZfp1 is associated with mycelial growth, conidiation, osmoregulation, and pathogenicity in F. oxysporum on P. kingianum.\u003c/span\u003e\u003c/p\u003e\u003cp\u003e\u003cem\u003ePolygonatum kingianum\u003c/em\u003e Coll. et Hemsl. is a native medicinal plant in Yunnan province, whose rhizome has medicinal and dietary concomitant functions\u0026nbsp;\u003csup\u003e1,2\u003c/sup\u003e. However, rhizome rot seriously threatens the sustainable production of \u003cem\u003eP. kingianum\u003c/em\u003e. The causal agents of rhizome rot in\u003cem\u003e\u0026nbsp;P. kingianum\u0026nbsp;\u003c/em\u003eare \u003cem\u003eFusarium oxysporum\u003c/em\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003eand\u003cem\u003e\u0026nbsp;F. solani,\u0026nbsp;\u003c/em\u003eand the former is more virulent\u0026nbsp;\u003csup\u003e3\u003c/sup\u003e\u003cem\u003e. F. oxysporum\u003c/em\u003e is a destructive soil-borne vascular fungal pathogen and the fifth largest plant pathogenic fungus globally that causes fusarium wilt, root rot, and necrosis, leading to severe yield losses in over 100 host plants\u0026nbsp;\u003csup\u003e4-6\u003c/sup\u003e. However, there are few effective control methods against\u0026nbsp;\u003cem\u003eF. oxysporum\u003c/em\u003e, given the long-term survival of its chlamydospores in the soil and mycelia colonization in host xylem vessels\u0026nbsp;\u003csup\u003e7\u003c/sup\u003e. Therefore, understanding the pathogenic mechanism of rhizome rot in\u003cem\u003e\u0026nbsp;P. kingianum\u003c/em\u003e and screening and identifying the \u003cem\u003eF. oxysporum\u003c/em\u003e pathogenic genes can provide clues for exploring the\u003cem\u003e\u0026nbsp;\u003c/em\u003escientific prevention and control measures against rhizome rot.\u003c/p\u003e\n\u003cp\u003eTranscription factors (TFs) regulate cell development, differentiation, and the cellular response to external\u0026nbsp;perturbation\u0026nbsp;by binding to a specific DNA site, or sites, where transcription activation or repression occurs through various mechanisms, including DNA\u0026ndash;protein interactions, protein\u0026ndash;protein interactions, and modification of the chromatin structure\u0026nbsp;\u003csup\u003e8-10\u003c/sup\u003e. The zinc finger (ZF) protein is a TF with a \u0026lsquo;finger\u0026rsquo; domain that regulates gene expression. It stabilizes a short polypeptide spatial configuration, folded into a finger-like structure by binding Zn\u003csup\u003e2+\u003c/sup\u003e. The ZF protein was first identified in \u003cem\u003eXenopus\u003c/em\u003e oocytes\u0026nbsp;\u003csup\u003e11,12\u003c/sup\u003e, and is widely distributed in animals, plants and microorganisms\u0026nbsp;\u003csup\u003e13\u003c/sup\u003e. It is divided into several subfamilies based on the numbers and positions of cysteine (Cys) and histidine (His) residues, including Cys\u003csub\u003e2\u003c/sub\u003e/His\u003csub\u003e2\u003c/sub\u003e-type (C\u003csub\u003e2\u003c/sub\u003eH\u003csub\u003e2\u003c/sub\u003e), C\u003csub\u003e2\u003c/sub\u003eHC, C\u003csub\u003e2\u003c/sub\u003eC\u003csub\u003e2\u003c/sub\u003e, C\u003csub\u003e2\u003c/sub\u003eHCC\u003csub\u003e2\u003c/sub\u003eC\u003csub\u003e2\u003c/sub\u003e, and C\u003csub\u003e2\u003c/sub\u003eC\u003csub\u003e2\u003c/sub\u003eC\u003csub\u003e2\u003c/sub\u003eC\u003csub\u003e2\u0026nbsp;\u003c/sub\u003e\u003csup\u003e14\u003c/sup\u003e. More than 700 TFs have been predicted in \u003cem\u003eF. oxysporum\u003c/em\u003e genome. However, only 26 TFs have been functionally analyzed, with the majority (15 TFs) belonging to the ZF protein family\u0026nbsp;\u003csup\u003e15\u003c/sup\u003e. The 15 TFs include six Zn(II)\u003csub\u003e2\u003c/sub\u003eCys\u003csub\u003e6\u003c/sub\u003e ZFs, five C\u003csub\u003e2\u003c/sub\u003eH\u003csub\u003e2\u003c/sub\u003e ZFs, two GATA ZFs, and two plant homeodomain (PHD)-containing ZFs\u0026nbsp;\u003csup\u003e15\u003c/sup\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe\u003cem\u003e\u0026nbsp;F. oxysporum\u003c/em\u003e homolog of the TF \u003cem\u003eSte12\u003c/em\u003e possesses a C\u003csub\u003e2\u003c/sub\u003eH\u003csub\u003e2\u0026nbsp;\u003c/sub\u003edomain, was up-regulated during the infection process, and was necessary for \u003cem\u003eF. oxysporum\u003c/em\u003e virulence\u0026nbsp;\u003csup\u003e16\u003c/sup\u003e. On the contrary, the pH signalling TF \u003cem\u003ePacC\u003c/em\u003e with a C\u003csub\u003e2\u003c/sub\u003eH\u003csub\u003e2\u0026nbsp;\u003c/sub\u003edomain negatively regulated virulence, preventing the transcription of acid-expressed genes essential during \u003cem\u003eF. oxysporum\u003c/em\u003e infection\u0026nbsp;\u003csup\u003e17\u003c/sup\u003e. Zinc homeostasis regulator \u003cem\u003eZafA\u0026nbsp;\u003c/em\u003eis also a C\u003csub\u003e2\u003c/sub\u003eH\u003csub\u003e2\u0026nbsp;\u003c/sub\u003eZF. It was significantly up-regulated during the early stages of infection and was required for the full virulence of \u003cem\u003eF. oxysporum,\u0026nbsp;\u003c/em\u003eespecially when zinc was limited\u0026nbsp;\u003csup\u003e18\u003c/sup\u003e. However, \u003cem\u003eFolCzf1\u003c/em\u003e, a C\u003csub\u003e2\u003c/sub\u003eH\u003csub\u003e2\u003c/sub\u003e ZF in \u003cem\u003eF. oxysporum\u003c/em\u003e f. sp. \u003cem\u003elycopersici\u003c/em\u003e (\u003cem\u003eFol\u003c/em\u003e), was required for growth, conidiation, conidia morphology, and pathogenicity in tomato\u0026nbsp;\u003csup\u003e19\u003c/sup\u003e. Similarly, the \u003cem\u003eCon7-1\u0026nbsp;\u003c/em\u003e(a C\u003csub\u003e2\u003c/sub\u003eH\u003csub\u003e2\u003c/sub\u003e ZF in \u003cem\u003eF. oxysporum\u003c/em\u003e)\u0026nbsp;deletion mutant exhibited defects in chitin synthase, hyphal branch, conidiation, and virulence\u0026nbsp;\u003csup\u003e20\u003c/sup\u003e. Although these five C\u003csub\u003e2\u003c/sub\u003eH\u003csub\u003e2\u003c/sub\u003e ZFs have been identified and functionally tested, this is not even close to sufficient.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTherefore, this study aimed to investigate the roles this new ZF protein TF \u003cem\u003eFoZfp1\u003c/em\u003e with two C\u003csub\u003e2\u003c/sub\u003eH\u003csub\u003e2\u0026nbsp;\u003c/sub\u003edomains in the developmental processes and pathogenicity of the \u003cem\u003eF. oxysporum\u0026nbsp;\u003c/em\u003ein\u003cem\u003e\u0026nbsp;P. kingianum\u003c/em\u003e. The \u003cem\u003eFoZfp1\u0026nbsp;\u003c/em\u003edeletion mutants\u0026nbsp;(\u003cem\u003e△\u003c/em\u003e\u003cem\u003eFoZfp1\u003c/em\u003e) and the mutant complementary (\u003cem\u003e△\u003c/em\u003e\u003cem\u003eFoZfp1\u003c/em\u003e-C) strains were generated by the target gene replacement technique.\u0026nbsp;The \u003cem\u003eFoZfp1\u003c/em\u003e was up-regulated during \u003cem\u003eF. oxysporum\u003c/em\u003e conidial germination\u003cem\u003e.\u003c/em\u003e The inhibition rates, sensitivity under cell wall and osmotic targeted stresses, and virulence of\u0026nbsp;\u003cem\u003e△\u003c/em\u003e\u003cem\u003eFoZfp1\u0026nbsp;\u003c/em\u003ewere decreased compared to those of WT and\u0026nbsp;\u003cem\u003e△\u003c/em\u003e\u003cem\u003eFoZfp1\u003c/em\u003e-C. The findings from this study elaborate on the effects of \u003cem\u003eFoZfp1\u003c/em\u003e on \u003cem\u003eF. oxysporum\u003c/em\u003e growth, conidiation, response against stress, and virulence\u003cem\u003e.\u003c/em\u003e\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eAssessment of \u003cem\u003eFoZfp1\u003c/em\u003e expression pattern during conidial germination\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTranscriptome analysis revealed that the\u003cem\u003e\u0026nbsp;FoZfp1\u0026nbsp;\u003c/em\u003eexpression was up-regulated in \u003cem\u003eF. oxysporum\u003c/em\u003e conidia cultured for 12 h and 24 h compared to the control (0 h) \u003csup\u003e21\u003c/sup\u003e\u003cem\u003e.\u003c/em\u003e The qRT-PCR results revealed that \u003cem\u003eFoZfp1\u0026nbsp;\u003c/em\u003ewas up-regulated during \u003cem\u003eF. oxysporum\u0026nbsp;\u003c/em\u003econidial germination (Fig. 1).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eFoZfp1\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;is a C\u003csub\u003e2\u003c/sub\u003eH\u003csub\u003e2\u003c/sub\u003e-type ZF protein\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBLASTn analysis revealed that \u003cem\u003eFoZfp1\u003c/em\u003e nucleotide sequence (OR715798) was 99% identical to \u003cem\u003eF. oxysporum\u003c/em\u003e f. sp. \u003cem\u003elycopersici\u003c/em\u003e 4287 ZF protein MSN2/4 (XM_018379112) \u003csup\u003e22\u003c/sup\u003e. Additionally, \u003cem\u003eFoZfp1\u003c/em\u003e was classified into the C\u003csub\u003e2\u003c/sub\u003eH\u003csub\u003e2\u003c/sub\u003e-type subfamily, \u003cem\u003eSte12\u003c/em\u003e (ACM80357) \u003csup\u003e16\u003c/sup\u003e, containing two C\u003csub\u003e2\u003c/sub\u003eH\u003csub\u003e2\u003c/sub\u003e zinc finger domains (Fig. 2A, B), making it a C\u003csub\u003e2\u003c/sub\u003eH\u003csub\u003e2\u003c/sub\u003e-type ZF protein.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eVerification\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;of \u003cem\u003eFoZfp1\u003c/em\u003e deletion and complementary mutants\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe hygromycin B-resistant transformants were verified by PCR. The \u003cem\u003eFoZfp1\u003c/em\u003e gene was successfully replaced in the \u003cem\u003eFoZfp1\u003c/em\u003e transformants (\u003cem\u003e△\u003c/em\u003e\u003cem\u003eFoZfp1\u003c/em\u003e) (Fig. 3A). Co-transformation of the \u003cem\u003eFoZfp1\u003c/em\u003e gene with the vector pDHtsk-GFP-G418 successfully complemented the\u003cem\u003e\u0026nbsp;FoZfp1\u003c/em\u003e gene (\u003cem\u003e△\u003c/em\u003e\u003cem\u003eFoZfp1\u003c/em\u003e-C). Additionally, the target \u003cem\u003eFoZfp1\u0026nbsp;\u003c/em\u003enucleotide sequences were amplified in WT and \u003cem\u003e△\u003c/em\u003e\u003cem\u003eFoZfp1\u003c/em\u003e-C but not in \u003cem\u003e△\u003c/em\u003e\u003cem\u003eFoZfp1\u0026nbsp;\u003c/em\u003e(Fig. 3B)\u003cem\u003e.\u0026nbsp;\u003c/em\u003eAt the same time, the hygromycin B-resistance gene were amplified in \u003cem\u003e△\u003c/em\u003e\u003cem\u003eFoZfp1\u003c/em\u003e and \u003cem\u003e△\u003c/em\u003e\u003cem\u003eFoZfp1\u003c/em\u003e-C but not in WT (Fig. 3C). Furthermore, the neomycin-resistance gene were amplified in \u003cem\u003e△\u003c/em\u003e\u003cem\u003eFoZfp1\u003c/em\u003e-C but not in WT and \u003cem\u003e△\u003c/em\u003e\u003cem\u003eFoZfp1\u0026nbsp;\u003c/em\u003e(Fig. 3D). Validation of the \u003cem\u003eFoZfp1\u003c/em\u003e expression levels by qRT-PCR revealed that \u003cem\u003e△\u003c/em\u003e\u003cem\u003eFoZfp1\u003c/em\u003e expression was significantly reduced compared to WT and \u003cem\u003e△\u003c/em\u003e\u003cem\u003eFoZfp1\u003c/em\u003e-C expressions (Fig. 3E)\u003cem\u003e.\u0026nbsp;\u003c/em\u003eThese results indicated that \u003cem\u003eFoZfp1\u0026nbsp;\u003c/em\u003ewas knocked out in \u003cem\u003e△\u003c/em\u003e\u003cem\u003eFoZfp1\u003c/em\u003e and was complemented in \u003cem\u003e△\u003c/em\u003e\u003cem\u003eFoZfp1\u003c/em\u003e-C.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeletion of\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003cem\u003eFoZfp1\u003c/em\u003e affects mycelial growth and conidial formation\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe \u003cem\u003e△\u003c/em\u003e\u003cem\u003eFoZfp1\u0026nbsp;\u003c/em\u003ecolony formation and growth rates were significantly slow and reduced compared to WT and \u003cem\u003e△\u003c/em\u003e\u003cem\u003eFoZfp1\u003c/em\u003e-C cultured on potato dextrose agar (PDA) for 5 days (Fig. 4A, B). Furthermore, a significantly reduced number of micro-conidia was produced in \u003cem\u003e△\u003c/em\u003e\u003cem\u003eFoZfp1\u003c/em\u003e at 96 h post-inoculation on potato dextrose broth (PDB) (Fig. 4C). However, \u003cem\u003e△\u003c/em\u003e\u003cem\u003eFoZfp1\u003c/em\u003e spore morphology was not different compared to WT and \u003cem\u003e△\u003c/em\u003e\u003cem\u003eFoZfp1\u003c/em\u003e-C (Fig. 4D). These results indicated that deletion of \u003cem\u003eFoZfp1\u003c/em\u003e affected mycelial growth and conidial formation in \u003cem\u003eF. oxysporum\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeletion of \u003cem\u003eFoZfp1\u003c/em\u003e impairs its pathogenicity\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn the infection assays, the \u003cem\u003e△\u003c/em\u003e\u003cem\u003eFoZfp1\u003c/em\u003e lesion diameters in the detached leaves and rhizomes were significantly decreased compared to WT and \u003cem\u003e△\u003c/em\u003e\u003cem\u003eFoZfp1\u003c/em\u003e-C (Fig. 5A, B). Withering of the leaves and rhizome rot were observed among the \u003cem\u003eP. kingianum\u003c/em\u003e plants inoculated with WT and \u003cem\u003e△\u003c/em\u003e\u003cem\u003eFoZfp1\u003c/em\u003e-C. However, no symptoms were observed in \u003cem\u003e△\u003c/em\u003e\u003cem\u003eFoZfp1\u003c/em\u003e (Fig. 5C-E). Overall, the disease index of WT and \u003cem\u003e△\u003c/em\u003e\u003cem\u003eFoZfp1\u003c/em\u003e-C was significantly higher than that of \u003cem\u003e△\u003c/em\u003e\u003cem\u003eFoZfp1\u0026nbsp;\u003c/em\u003e(Fig. 5F)\u003cem\u003e.\u0026nbsp;\u003c/em\u003eThese results demonstrated that \u003cem\u003eFoZfp1\u003c/em\u003e was required for full virulence in \u003cem\u003eF. oxysporum\u003c/em\u003e on \u003cem\u003eP. kingianum.\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eFoZfp1\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;contributes to \u003cem\u003eF. oxysporum\u003c/em\u003e stress responses\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe inhibition rates\u0026nbsp;and sensitivity of\u0026nbsp;\u003cem\u003e△\u003c/em\u003e\u003cem\u003eFoZfp1\u003c/em\u003e under cell wall (sodium dodecyl sulfate, SDS; congo red, CR), osmotic stress (NaCl, KCl), and tebuconazole stresses were decreased compared to WT and\u0026nbsp;\u003cem\u003e△\u003c/em\u003e\u003cem\u003eFoZfp1\u003c/em\u003e-C.\u0026nbsp;However,\u0026nbsp;\u003cem\u003e△\u003c/em\u003e\u003cem\u003eFoZfp1\u003c/em\u003e had a lower tolerance to carbendazim compared to WT and\u0026nbsp;\u003cem\u003e△\u003c/em\u003e\u003cem\u003eFoZfp1\u003c/em\u003e-C. Besides, the differences in the inhibition rates on\u0026nbsp;\u003cem\u003e△\u003c/em\u003e\u003cem\u003eFoZfp1,\u003c/em\u003e WT, and\u0026nbsp;\u003cem\u003e△\u003c/em\u003e\u003cem\u003eFoZfp1\u003c/em\u003e-C were insignificant under oxidative stress (H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e), suggesting that \u003cem\u003eFoZfp1\u003c/em\u003e had little effect on the sensitivity to oxidative stress (Fig. 6). These results suggested that \u003cem\u003eFoZfp1\u003c/em\u003e was involved in regulating responses to osmotic pressure and cell wall integrity stresses in \u003cem\u003eF. oxysporum\u003c/em\u003e.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eRhizome rot is a devastating soil-borne disease, seriously threatening the \u003cem\u003eP. kingianum\u003c/em\u003e industry. \u003cem\u003eF. oxysporum\u0026nbsp;\u003c/em\u003einvades the roots, causing wounds, colonizes the vascular tissues, blocking water and nutrient transport\u003cem\u003e,\u003c/em\u003e and may even lead to plant death\u0026nbsp;\u003csup\u003e23\u003c/sup\u003e\u003cem\u003e.\u0026nbsp;\u003c/em\u003eAlthough over 700 TFs have been predicted in \u003cem\u003eF. oxysporum\u003c/em\u003e, only 15 ZFs are associated with pathogenicity\u0026nbsp;\u003csup\u003e15\u003c/sup\u003e. Among them is \u003cem\u003eFow2\u003c/em\u003e, a Zn(II)\u003csub\u003e2\u003c/sub\u003eCys\u003csub\u003e6\u003c/sub\u003e-type transcription regulator, essential for root invasion and colonization but not for vegetative growth and conidiation in \u003cem\u003eF. oxysporum\u0026nbsp;\u003c/em\u003e\u003csup\u003e24\u003c/sup\u003e. The cutinase transcription factors \u003cem\u003ectf1\u003c/em\u003e and \u003cem\u003ectf2\u003c/em\u003e\u003cem\u003e,\u0026nbsp;\u003c/em\u003econtaining\u003cem\u003e\u0026nbsp;\u003c/em\u003ethe\u003cem\u003e\u0026nbsp;\u003c/em\u003eZn\u003csub\u003e2\u003c/sub\u003eCys\u003csub\u003e6\u003c/sub\u003e DNA binding domain,\u003cem\u003e\u0026nbsp;\u003c/em\u003ealso\u003cem\u003e\u0026nbsp;\u003c/em\u003eplay important roles in the lipolytic system\u003cem\u003e\u0026nbsp;\u003c/em\u003eof \u003cem\u003eFol\u003c/em\u003e. Additionally, \u003cem\u003ectf1\u003c/em\u003e and \u003cem\u003ectf2\u003c/em\u003e deletion mutants severely reduce \u003cem\u003eF. oxysporum\u003c/em\u003e virulence\u0026nbsp;\u003csup\u003e25\u003c/sup\u003e. In common bean, \u003cem\u003eFusarium\u003c/em\u003e transcription factor 1 (\u003cem\u003eftf1\u003c/em\u003e) with a Zn(II)\u003csub\u003e2\u003c/sub\u003eCys\u003csub\u003e6\u003c/sub\u003e motif is only up-regulated during plant infection, where multiple \u003cem\u003eftf1\u003c/em\u003e copies increase virulence in \u003cem\u003eF. oxysporum\u003c/em\u003e f. sp. \u003cem\u003ephaseol\u0026nbsp;\u003c/em\u003e\u003csup\u003e26\u003c/sup\u003e. Additionally, \u003cem\u003eEBR1\u003c/em\u003e, belonging to the Zn\u003csub\u003e2\u003c/sub\u003eCys\u003csub\u003e6\u003c/sub\u003e family, regulates the expression of genes encoding metabolism and virulence.\u003cem\u003e\u0026nbsp;EBR1\u003c/em\u003e deletion impairs growth and reduces pathogenicity and biocontrol capacities in different \u003cem\u003eF. oxysporum\u003c/em\u003e strains\u0026nbsp;\u003csup\u003e27\u003c/sup\u003e. Furthermore, the global nitrogen regulator\u003cem\u003e\u0026nbsp;FNR1\u003c/em\u003e, with a single conserved GATA-type ZF domain, regulates the secondary nitrogen acquisition in plants. Notably, the disruption of \u003cem\u003eFNR1\u0026nbsp;\u003c/em\u003emutants significantly delays \u003cem\u003eF. oxysporum\u003c/em\u003e infection in tomato seedlings\u0026nbsp;\u003csup\u003e28\u003c/sup\u003e. Besides, \u003cem\u003eCti6\u003c/em\u003e, which contains a PHD finger motif and simultaneously interacts with the transcriptional corepressor complex Cyc8-Tup1 and the co-activator SAGA (Spt-Ada-Gcn5-acetytransferase) complex, is required for full virulence in \u003cem\u003eF. oxysporum\u003c/em\u003e on tomato\u0026nbsp;\u003csup\u003e29\u003c/sup\u003e. In this study, C\u003csub\u003e2\u003c/sub\u003eH\u003csub\u003e2\u003c/sub\u003e ZF \u003cem\u003eFoZfp1\u0026nbsp;\u003c/em\u003ewas identified, and it was required for full virulence in \u003cem\u003eF. oxysporum\u003c/em\u003e on\u003cem\u003e\u0026nbsp;P. kingianum\u003c/em\u003e. However, the regulatory mechanism in pathogenicity needs to be analyzed in the plant-pathogen interaction.\u003c/p\u003e\n\u003cp\u003eThe host immune system first detects the pathogenic fungi conidia during the infection process. With \u003cem\u003eF. oxysporum\u003c/em\u003e, conidial germination is the key step of infection\u0026nbsp;\u003csup\u003e30\u003c/sup\u003e.\u0026nbsp;Therefore, early conidial detection is crucial to inhibit fungal growth and alleviate the disease occurrence\u0026nbsp;\u003csup\u003e31\u003c/sup\u003e. In this study, C\u003csub\u003e2\u003c/sub\u003eH\u003csub\u003e2\u003c/sub\u003e ZF \u003cem\u003eFoZfp1\u003c/em\u003e was screened using the genome and transcriptome data of the conidial germination process in \u003cem\u003eF. oxysporum\u003c/em\u003e. \u003cem\u003eFoZfp1\u003c/em\u003e was significantly up-regulated during conidial germination, suggesting that this gene might be related to \u003cem\u003eF. oxysporum\u003c/em\u003e growth and pathogenicity. To verify this conjecture, \u003cem\u003eFoZfp1\u003c/em\u003e and the mutant complementary strains were constructed using split-marker homologous recombination, which revealed that \u003cem\u003eFoZfp1\u003c/em\u003e regulated mycelial growth and conidial yield. However, this did not affect the conidia morphology. Similarly, \u003cem\u003eBcTaf14\u003c/em\u003e, TATA box-binding protein-associated factor 14 (Taf14) in \u003cem\u003eBotrytis cinerea\u003c/em\u003e was associated with mycelial growth and conidial morphogenesis with no effect on conidial germination ratio and rate\u0026nbsp;\u003csup\u003e32\u003c/sup\u003e. \u003cem\u003esnt2\u003c/em\u003e, a PHD-containing ZF, was also essential in vegetative growth, conidial production, and host colonization by \u003cem\u003eF. oxysporum\u003c/em\u003e f. sp. \u003cem\u003emelonis\u003c/em\u003e \u003csup\u003e33\u003c/sup\u003e. However, the white-collar 1 photoreceptor \u003cem\u003eWc1\u003c/em\u003e,\u003cem\u003e\u0026nbsp;\u003c/em\u003ea GATA ZF, and knockout mutants of \u003cem\u003eF. oxysporum\u0026nbsp;\u003c/em\u003elacking\u003cem\u003e\u0026nbsp;Wc1\u0026nbsp;\u003c/em\u003eimpaired aerial hyphae and virulence\u0026nbsp;\u003csup\u003e34\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eWhen a pathogen invades the host, the host exhibits some defensive response, including a change in the internal environment or vascular system\u0026nbsp;\u003csup\u003e35\u003c/sup\u003e. Similarly, pathogenic fungi always adopt a series of complex strategies for successful invasion of the host, including the plant defense mechanisms, host intracellular environment, and defeating adverse environmental changes\u0026nbsp;\u003csup\u003e36,37\u003c/sup\u003e. In this study,\u0026nbsp;\u003cem\u003e△\u003c/em\u003e\u003cem\u003eFoZfp1\u0026nbsp;\u003c/em\u003eincreased resistance to membrane stressor (SDS), cell wall stressor (CR), extracellular osmotic (NaCl), and intracellular osmotic (KCl) stress compared to the WT strain. However, no differences were identified in tolerance to oxidative (H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e) stress between the mutants and the WT strains, implying that \u003cem\u003eFoZfp1\u003c/em\u003e regulated osmotic pressure and cell wall integrity stresses. These results are similar to those of \u003cem\u003eAoime2\u003c/em\u003e (\u003cem\u003eArthrobotrys oligospora\u003c/em\u003e inducer of meiosis 2) deletion mutants, unaffected by oxidative stressor H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e but highly sensitive to the osmotic stressor NaCl\u0026nbsp;\u003csup\u003e38\u003c/sup\u003e. Similarly, \u003cem\u003eBcTaf14\u003c/em\u003e deletion mutants increased NaCl and KCl sensitivity\u0026nbsp;\u003csup\u003e32\u003c/sup\u003e. Neutral trehalase-encoding gene \u003cem\u003eNTH1\u003c/em\u003e knockout mutant was also sensitive to H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u0026nbsp;\u003c/sub\u003eand SDS but not to CR, NaCl, and KCl\u0026nbsp;\u003csup\u003e39\u003c/sup\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn conclusion, the C\u003csub\u003e2\u003c/sub\u003eH\u003csub\u003e2\u003c/sub\u003e ZF-encoding gene \u003cem\u003eFoZfp1\u003c/em\u003e plays significant roles in mycelial growth, conidiation, stress response, and pathogenicity in \u003cem\u003eF. oxysporum\u0026nbsp;\u003c/em\u003eof \u003cem\u003eP. kingianum.\u003c/em\u003e However, how \u003cem\u003eFoZfp1\u003c/em\u003e exerts these functions requires further study.\u003c/p\u003e"},{"header":"Materials And Methods","content":"\u003cp\u003e\u003cstrong\u003eIsolation of the fungal strain and the culture conditions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe wild-type\u003cem\u003e\u0026nbsp;F. oxysporum\u0026nbsp;\u003c/em\u003estrain PkF01 isolated from the \u003cem\u003eP. kingianum\u003c/em\u003e rhizome rot samples, was identified with nucleotide sequences of the elongation factor 1-alpha (MW149127) and the second largest subunit of nuclear DNA-directed RNA polymerase II (MW194100) by L. Zhang in our previous study \u003csup\u003e3\u003c/sup\u003e. For conidia production, mycelia were incubated in PDB at 28\u0026deg;C with shaking at 180 revolutions per minute (rpm) for 3 days. Subsequently, the conidial suspension was adjusted to 1\u0026times;10\u003csup\u003e6\u0026nbsp;\u003c/sup\u003econidia\u0026middot;mL\u003csup\u003e-1\u003c/sup\u003e, and 30% glycerol was added before storing the suspension at \u0026minus;80\u0026deg;C \u003csup\u003e40\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePhylogenetic tree construction and protein sequence alignment\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eFoZfp1\u003c/em\u003e nucleotide sequence was submitted to the National Center for Biotechnology Information (NCBI) GenBank database, and BLASTn analysis was performed in NCBI. To further investigate the function of \u003cem\u003eFoZfp1\u003c/em\u003e in \u003cem\u003eF. oxysporum\u003c/em\u003e, phylogenetic tree and alignment of the C\u003csub\u003e2\u003c/sub\u003eH\u003csub\u003e2\u003c/sub\u003e zinc-finger proteins of \u003cem\u003eF. oxysporum\u003c/em\u003e were performed using the maximum likelihood method with 1,000 replications of bootstrap in MEGA 11 \u003csup\u003e41\u003c/sup\u003e and edited in GeneDoc \u003csup\u003e42\u003c/sup\u003e, respectively.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGeneration of \u003cem\u003eFoZfp1\u003c/em\u003e deletion mutants\u003cem\u003e\u0026nbsp;\u003c/em\u003e(\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003e△\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003eFoZfp1\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe \u003cem\u003eFoZfp1\u003c/em\u003e gene in WT was deleted using the split-marker recombination technology \u003csup\u003e43,44\u003c/sup\u003e. Firstly, 658 bp\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eupstream (\u003cem\u003eFoZfp1\u003c/em\u003e-Up)\u003cem\u003e\u0026nbsp;\u003c/em\u003eand\u003cem\u003e\u0026nbsp;\u003c/em\u003e807 bp\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003edownstream (\u003cem\u003eFoZfp1\u003c/em\u003e-Down) \u003cem\u003eFoZfp1\u003c/em\u003e fragments, and 800 bp upstream (Hy) and 1,112 bp downstream (Yg) hygromycin B-resistance cassette (HYG) fragments from the vector, pZD101-AmCyan \u003csup\u003e44\u003c/sup\u003e were amplified using four sets of primers \u003cem\u003eFoZfp1\u003c/em\u003e-UF/\u003cem\u003eFoZfp1\u003c/em\u003e-UR,\u003cem\u003e\u0026nbsp;FoZfp1\u003c/em\u003e-DF/\u003cem\u003eFoZfp1\u003c/em\u003e-DR, Hy-F/Hy-R,\u003cem\u003e\u0026nbsp;\u003c/em\u003eand Yg-F/Yg-R, respectively. Secondly, \u003cem\u003eFoZfp1\u003c/em\u003e-Up and Hy were fused through PCR splicing by overlap extension \u003csup\u003e45\u003c/sup\u003e, using the \u003cem\u003eFoZfp1\u003c/em\u003e-UF/Hy-R primer pair and \u003cem\u003eFoZfp1\u003c/em\u003e-Up/Hy as the templates. Additionally, \u003cem\u003eFoZfp1\u003c/em\u003e-Down was fused with Yg using the Yg-F/\u003cem\u003eFoZfp1\u003c/em\u003e-DR primer pair and \u003cem\u003eFoZfp1\u003c/em\u003e-Down/Yg as the template. Thirdly, the two fusion fragments were transformed into WT protoplasts following the polyethylene glycol-mediated protoplast transformation technique \u003csup\u003e46\u003c/sup\u003e. Finally, the transformants were screened on PDA containing ampicillin (100 mg\u0026middot;L\u003csup\u003e-1\u003c/sup\u003e) and hygromycin B (400 mg\u0026middot;L\u003csup\u003e-1\u003c/sup\u003e).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eComplementation of \u003cem\u003eFoZfp1\u003c/em\u003e deletion mutant\u003cem\u003e\u0026nbsp;\u003c/em\u003e(\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003e△\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003eFoZfp1-C\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e△\u003c/em\u003e\u003cem\u003eFoZfp1\u003c/em\u003e was complemented with a fragment containing \u003cem\u003eFoZfp1\u0026nbsp;\u003c/em\u003efull-length cDNA. The fragment amplified with \u003cem\u003eFoZfp1\u003c/em\u003e-CF/\u003cem\u003eFoZfp1\u003c/em\u003e\u003cem\u003e-\u003c/em\u003eCR primer pair was cloned into the T3 promoter vector pDHtsk-GFP-G418 with a neomycin-resistant cassette. Next, the recombinant plasmid was transformed into \u003cem\u003e△\u003c/em\u003e\u003cem\u003eFoZfp1\u003c/em\u003e protoplasts. Subsequently, the transformants were screened on PDA containing hygromycin B (400 mg\u0026middot;L\u003csup\u003e-1\u003c/sup\u003e) and neomycin (300 mg\u0026middot;L\u003csup\u003e-1\u003c/sup\u003e).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eVerification and quantification of gene expression\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe deletion mutants were verified by PCR using primer pair \u003cem\u003eFoZfp1\u003c/em\u003e-IF/\u003cem\u003eFoZfp1\u003c/em\u003e-IF,\u003cem\u003e\u0026nbsp;FoZfp1\u003c/em\u003e-UH-F/\u003cem\u003eFoZfp1\u003c/em\u003e-UH-R,\u003cem\u003e\u0026nbsp;\u003c/em\u003eand \u003cem\u003eFoZfp1-\u003c/em\u003eDY-F\u003cem\u003e/FoZfp1-\u003c/em\u003eDY-R. The complementary mutants were verified using \u003cem\u003eFoZfp1\u003c/em\u003e-IF/\u003cem\u003eFoZfp1\u003c/em\u003e-IR, \u003cem\u003eHy\u003c/em\u003e-F\u003cem\u003e/Yg\u003c/em\u003e-R, and \u003cem\u003eNeo\u003c/em\u003e-F\u003cem\u003e/Neo\u003c/em\u003e-R primer pairs. qRT-PCR further validated\u003cem\u003e\u0026nbsp;foZfp1\u0026nbsp;\u003c/em\u003eexpression. RNA extraction, cDNA synthesis, and qRT-PCR were performed with TaKaRa MiniBEST Universal RNA Extraction Kit (TaKaRa, Code No. 9767), PrimeScript\u0026trade; RT Master Mix (TaKaRa, Code No. RR036A), and TB Green\u0026reg; Premix Ex Taq\u0026trade; II (TaKaRa, Code No. RR820A) according to manufacturer\u0026apos;s instructions, respectively. The qRT-PCR conditions were as follows: initial denaturation at 95\u0026deg;C for 30s, followed by 40 cycles of 95\u0026deg;C for 5 s, and annealing at 60\u0026deg;C for 30 s. Elongation factor 1-alpha (\u003cem\u003eEF1\u0026alpha;\u003c/em\u003e) and tubulin 2 (\u003cem\u003eTUB2\u003c/em\u003e) were used as internal reference genes \u003csup\u003e47\u003c/sup\u003e. The relative expression of the target gene was calculated by the 2\u003csup\u003e\u0026ndash;\u003c/sup\u003e\u003csup\u003e△△\u003c/sup\u003e\u003csup\u003eCt\u003c/sup\u003e method \u003csup\u003e48\u003c/sup\u003e. The qRT-PCR assay was conducted with three independent biological replicates. The \u003cem\u003eFoZfp1\u0026nbsp;\u003c/em\u003egene expression were quantified by qRT-PCR using primer pair \u003cem\u003eFoZfp1\u003c/em\u003e-QF1/\u003cem\u003eFoZfp1\u003c/em\u003e-QR1, \u003cem\u003eEF1\u0026alpha;\u003c/em\u003e-QF/\u003cem\u003eEF1\u0026alpha;\u003c/em\u003e-QR, and \u003cem\u003eTUB2\u003c/em\u003e-QF/\u003cem\u003eTUB2\u003c/em\u003e-QR. All primers used in this study were listed in Table S1.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMycelial growth\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;and conidiation assays\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor mycelial growth, a 5-mm-diameter mycelial plug\u0026nbsp;from a 3-day-old culture was placed on PDA and incubated at 28\u0026deg;C for 5 days. For 5 days, the colony morphology was photographed, and the colony diameter was measured daily. For conidiation, a 5-mm-diameter mycelial plug from a 3-day-old culture was placed on PDB (200 mL) with shaking at 180 rpm and 28\u0026deg;C. The conidial yield was calculated at 48, 72, and 96 h post-inoculation on PDB using a blood counting chamber. Subsequently, conidia were filtered through two layers of lens paper and resuspended to a concentration of 1\u0026times;10\u003csup\u003e6\u003c/sup\u003e spores\u0026middot;mL\u003csup\u003e-1\u003c/sup\u003e in PDB. The conidia germination rate was calculated at 12 h post resuspension. The assays were performed with three biological and three technical replicates.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePathogenicity assays\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eP. Kingianum\u0026nbsp;\u003c/em\u003eplants collected from plantation in Kunming city of Yunnan province, China, and were permitted and identified by P. Ji from Institute of Medicinal Plant Cultivation. One drop of conidial suspension (1\u0026times;10\u003csup\u003e6\u003c/sup\u003e spores\u0026middot;mL\u003csup\u003e-1\u003c/sup\u003e) was dripped onto the surface of\u003cem\u003e\u0026nbsp;\u003c/em\u003eeach\u0026nbsp;\u003cem\u003eP. kingianum\u003c/em\u003e leaf and tuber\u0026nbsp;\u003csup\u003e49,50\u003c/sup\u003e. Leaves/rhizomes inoculated with sterile water were used as the controls. Inoculated leaves and rhizomes were cultured on moist filter paper at 28\u0026deg;C and 16-h light/8-h dark. Ten leaves and rhizomes were used for each treatment, with three biological replicates. Lesion diameters were measured at 5 days post-inoculation.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eMoreover, the roots of one-year-old\u003cem\u003e\u0026nbsp;P. kingianum\u003c/em\u003e plants were dipped into conidial suspension (1\u0026times;10\u003csup\u003e6\u003c/sup\u003e spores\u0026middot;mL\u003csup\u003e-1\u003c/sup\u003e) for 30 min. Plants whose roots were dipped in sterile water were used as the control. Subsequently, the treatment and control plants were transplanted in pots filled with sterile soil and maintained in a growth chamber at 28\u0026deg;C, 60% relative humidity, and 16-h light/8-h dark for 30 d. Disease index was calculated using the formula: [\u0026sum;(grade \u0026times; number of plants corresponding grade) / (4 \u0026times; total number of plants investigated )] \u0026times; 100. Grade: 0 = healthy plants; 1 = yellowing of the lower leaves; 2 = yellowing of upper leaves; 3 = yellowing of most of the leaves; 4 = severe wilting or plant death \u003csup\u003e51\u003c/sup\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eExperimental studies on plant samples, including the supply of plant material, comply with institutional, national and international guidelines and legislation\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResponse against stress\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA 5-mm-diameter mycelial plug from a 3-day-old culture was placed on PDA supplemented with 1.8 M NaCl, 1.8 M KCl, 0.05% SDS, 0.1% CR, 0.08% H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, 0.25 \u0026mu;g\u0026middot;mL\u003csup\u003e-1\u003c/sup\u003e tebuconazole, and 0.4 \u0026mu;g\u0026middot;mL\u003csup\u003e-1\u003c/sup\u003e carbendazim. All the cultures were incubated in the dark at 28\u0026deg;C. Subsequently, their colony diameter was measured after 5 days. The inhibition ratio (%) was calculated as (C-N)/C \u0026times; 100 \u003csup\u003e52\u003c/sup\u003e, where C is the colony diameter of control and N is the colony diameter of the treatment. All treatments and the control had three biological and technical replicates.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe analysis of variance (ANOVA) was performed by IBM SPSS Statistics 26 (IBM Corporation, USA). Significance in all the comparisons among means with standard deviation was calculated by ANOVA with Duncan\u0026rsquo;s multiple comparison adjustment. Diagrams were made by GraphPad Prism 8 (GraphPad Prism Software Inc., San Diego, CA).\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthors contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConceptualization, L.Z; Data curation, J. S.; Formal analysis, J.W., J.T., J.L., X.D. and J.D.; Funding acquisition, L.Z.; Methodology, J.W., J.T. and W.Y.; Project administration, L.Z.; Writing \u0026ndash; original draft, J.S.; Writing \u0026ndash; review \u0026amp; editing, X.C., P.J. and L.Z. All authors have read and approved the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was funded by National Natural Science Foundation of China (82360746), Yunnan Fundamental Research Projects (202101AT070245), Yunnan Provincial Science and Technology Department-Applied Basic Research Joint Special Funds of Yunnan University of Chinese Medicine (202101AZ070001-054), Academician (Expert) Workstation Project, Wang Yuan Chao Expert Workstation in Yunnan Province (202305AF150018), Yunnan Science and Technology Talent and Platform Program (202105AG070012), and Team Project of College of Chinese Materia Medica, Yunnan University of Chinese Medicine.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets generated and/or analysed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAdditional information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplementary Information\u0026nbsp;\u003c/strong\u003eTable S1 List of primers used in this study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCorrespondence\u0026nbsp;\u003c/strong\u003eand requests for materials should be addressed to L.Z.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eZhao, P. \u003cem\u003eet al.\u003c/em\u003e The genus Polygonatum: A review of ethnopharmacology, phytochemistry and pharmacology. 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Microbiol. 204, 455. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s00203-022-02964-0\u003c/span\u003e\u003cspan address=\"10.1007/s00203-022-02964-0\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2022).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-3899586/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3899586/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"Polygonatum kingianum rhizome rot is a destructive soil-borne disease caused by Fusarium oxysporum, which adversely affects the yield and sustainable development of P. kingianum. However, there are few effective control measures against rhizome rot. Thus, understanding the infection mechanism of F. oxysporum is essential to manage rhizome rot in P. kingianum effectively. In this study, zinc finger transcription factor FoZfp1 consisting of two C2H2 motifs was up-regulated during F. oxysporum conidial germination. The FoZfp1 gene deletion mutant (△FoZfp1) and the mutant complementary (△FoZfp1-C) strains were generated by the target gene replacement technique. Biological characteristic analyses revealed that the △FoZfp1 mycelial growth and conidial production were slower than those of the wild-type F. oxysporum (WT) and △FoZfp1-C. Additionally, the inhibition rates and sensitivity of △FoZfp1 under cell wall and osmotic targeted stresses were decreased compared to those of WT and △FoZfp1-C. Pathogenicity assays further revealed that the virulence of △FoZfp1 on the P. kingianum leaves and rhizomes was significantly reduced. These results indicate that FoZfp1 is associated with mycelial growth, conidiation, osmoregulation, and pathogenicity in F. oxysporum on P. kingianum.","manuscriptTitle":"Zinc finger transcription factor FoZfp1 is required for growth, conidiation, osmoregulation, and full virulence in the Polygonatum kingianum pathogen Fusarium oxysporum","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-02-16 14:44:37","doi":"10.21203/rs.3.rs-3899586/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-03-26T07:20:21+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-03-19T17:46:12+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"21706121-f211-40e0-92ed-874ff08029ba","date":"2024-02-26T19:06:24+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"90e0b772-ea5c-48a6-a72b-d14aa75ab101","date":"2024-02-24T21:17:59+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-02-22T16:49:26+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-02-17T17:14:23+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2024-02-14T10:33:27+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-02-14T10:27:05+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2024-01-26T09:38:04+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"6cbec1a7-765a-449a-939d-06e5fb574e0b","owner":[],"postedDate":"February 16th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":28757870,"name":"Biological sciences/Microbiology/Fungi/Fungal biology"},{"id":28757871,"name":"Biological sciences/Microbiology/Fungi/Fungal genetics"},{"id":28757872,"name":"Biological sciences/Microbiology/Fungi/Fungal pathogenesis"}],"tags":[],"updatedAt":"2024-07-08T06:07:33+00:00","versionOfRecord":[],"versionCreatedAt":"2024-02-16 14:44:37","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3899586","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3899586","identity":"rs-3899586","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}