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

doi:10.21203/rs.3.rs-3899586/v1

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

https://doi.org/10.21203/rs.3.rs-3899586/v1

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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

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 Zn²⁺. 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 Cys₂/His₂-type (C₂H₂), C₂HC, C₂C₂, C₂HCC₂C₂, and C₂C₂C₂C₂¹⁴. 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)₂Cys₆ ZFs, five C₂H₂ ZFs, two GATA ZFs, and two plant homeodomain (PHD)-containing ZFs ¹⁵.

The F. oxysporum homolog of the TF Ste12 possesses a C₂H₂domain, 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 C₂H₂domain negatively regulated virulence, preventing the transcription of acid-expressed genes essential during F. oxysporum infection ¹⁷. Zinc homeostasis regulator ZafA is also a C₂H₂ZF. 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 C₂H₂ 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 C₂H₂ ZF in F. oxysporum) deletion mutant exhibited defects in chitin synthase, hyphal branch, conidiation, and virulence ²⁰. Although these five C₂H₂ 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 C₂H₂domains 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.

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 C₂H₂-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 C₂H₂-type subfamily, Ste12 (ACM80357) ¹⁶, containing two C₂H₂ zinc finger domains (Fig. 2A, B), making it a C₂H₂-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 (H₂O₂), 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.

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)₂Cys₆-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 Zn₂Cys₆ 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)₂Cys₆ 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 Zn₂Cys₆ 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, C₂H₂ 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, C₂H₂ 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 (H₂O₂) 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 H₂O₂ 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 H₂O₂and SDS but not to CR, NaCl, and KCl ³⁹.

In conclusion, the C₂H₂ 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.

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 1×10⁶conidia·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 C₂H₂ 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 1×10⁶ 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 (1×10⁶ 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 (1×10⁶ 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% H₂O₂, 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).

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.

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No competing interests reported.

TableS1ListofprimersusedinthisstudySR.docx
Table S1 List of primers used in this study.

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

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