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. 2022 Aug;23(8):1122-1140.
doi: 10.1111/mpp.13213. Epub 2022 Apr 1.

A secreted ribonuclease effector from Verticillium dahliae localizes in the plant nucleus to modulate host immunity

Chun-Mei Yin  1   2 , Jun-Jiao Li  1 , Dan Wang  1 , Dan-Dan Zhang  1 , Jian Song  1 , Zhi-Qiang Kong  1 , Bao-Li Wang  1 , Xiao-Ping Hu  3 , Steven J Klosterman  4 , Krishna V Subbarao  5 , Jie-Yin Chen  1 , Xiao-Feng Dai  1   2
Affiliations

A secreted ribonuclease effector from Verticillium dahliae localizes in the plant nucleus to modulate host immunity

Chun-Mei Yin et al. Mol Plant Pathol. 2022 Aug.

Abstract

The arms race between fungal pathogens and plant hosts involves recognition of fungal effectors to induce host immunity. Although various fungal effectors have been identified, the effector functions of ribonucleases are largely unknown. Herein, we identified a ribonuclease secreted by Verticillium dahliae (VdRTX1) that translocates into the plant nucleus to modulate immunity. The activity of VdRTX1 causes hypersensitive response (HR)-related cell death in Nicotiana benthamiana and cotton. VdRTX1 possesses a signal peptide but is unlikely to be an apoplastic effector because its nuclear localization in the plant is necessary for cell death induction. Knockout of VdRTX1 significantly enhanced V. dahliae virulence on tobacco while V. dahliae employs the known suppressor VdCBM1 to escape the immunity induced by VdRTX1. VdRTX1 homologs are widely distributed in fungi but transient expression of 24 homologs from other fungi did not yield cell death induction, suggesting that this function is specific to the VdRTX1 in V. dahliae. Expression of site-directed mutants of VdRTX1 in N. benthamiana leaves revealed conserved ligand-binding sites that are important for VdRTX1 function in inducing cell death. Thus, VdRTX1 functions as a unique HR-inducing effector in V. dahliae that contributes to the activation of plant immunity.

Keywords: Verticillium dahliae; VdRTX1; host immunity; secreted ribonuclease.

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Conflict of interest statement

The authors declare that they have no competing interests.

Figures

FIGURE 1
FIGURE 1
VdRTX1 from Verticillium dahliae encodes a ribonuclease that induces cell death. (a) Schematics of the three secreted ribonucleases in V. dahliae and their domain structures. SP, signal peptide. (b) Phylogenetic relationship of fungal ribotoxin and nontoxic RNase protein sequences, as well as three secreted ribonuclease proteins identified in this study. The phylogenetic tree was prepared using MEGA 7.0 using maximum‐likelihood algorithm (Kumar et al., 2016). (c) Cell‐death‐induction assays using VdRTX1, VdRTX2, and VdRTX3 in Nicotiana benthamiana 4‐week‐old plants were conducted at 5 days following agroinfiltration. Bcl‐2‐associated X protein (BAX) and green fluorescent protein (GFP) were used as positive and negative controls, respectively. (d) Immunoblotting analysis in N. benthamiana leaves transiently expressing the proteins VdRTX1, VdRTX2, and VdRTX3. Ponceau S‐stained RuBisCO protein was used as a total protein loading control
FIGURE 2
FIGURE 2
VdRTX1 triggers immunity in Nicotiana benthamiana that is dependent on its ribonuclease activity. (a) Analysis of RNA degradation. Degradation of the plant ribosomal RNA was determined following incubation with the total proteins extracted from N. benthamiana leaves after transient expression of VdRTX1 for 48 h. N. benthamiana leaves injected with the cell‐death‐inducer VdSCP126 or sterile water were used as positive and negative controls, respectively. RNase A was used as a control to demonstrate degradation of rRNA. The integrity of RNA of the main peaks of 18S rRNA (green triangle box) and 28S rRNA (red triangle box) was detected by the LabChip GX Touch HT Nucleic Acid Analyzer (Perkin Elmer). (b) Identification of callose deposition in N. benthamiana leaves transiently expressing VdRTX1. Callose deposition in N. benthamiana leaves from 4‐week‐old plants was detected 2 days after transient expression of VdRTX1; leaves were stained with aniline blue. Green fluorescent protein (GFP) expression served as a control. Bars = 200 µm. (c) Reactive oxygen species (ROS) accumulation following transient expression of VdRTX1 in N. benthamiana leaves. ROS accumulation in N. benthamiana leaves from 4‐week‐old plants was assessed 2 days after transient expression of VdRTX1; ROS were detected using 3,3′‐diaminobenzidine (DAB) staining. GFP expression served as a control. (d) Quantitative analysis of DAB staining results of N. benthamiana leaves by ImageJ. Data represent mean densities of DAB‐stained area from three leaves. Statistical significance was calculated using a Student's t test. Asterisks *** indicate significant differences (p < 0.001). (e) Relative transcript levels of immunity‐related genes in N. benthamiana following VdRTX1 transient expression. N. benthamiana leaves were sampled for RNA extraction at 2 days after agroinfiltration with A. tumefaciens expressing VdRTX1 or GFP as a control. Transcript levels of candidate genes were normalized to the levels of the reference gene NbEF1α and calibrated to the level of the GFP control expression (set as 1) according to the 2−ΔΔ C t method. Data represent means and SE (n = 12) from three independent biological replicates. Statistical significance was calculated using a Student's t test. Asterisks indicate significant differences (***p < 0.001, **p < 0.01)
FIGURE 3
FIGURE 3
VdRTX1 functions intracellularly and independently from immune receptors of Nicotiana benthamiana. (a) Secretory activity assays of the predicted signal peptide of VdRTX1 from Verticillium dahliae using the yeast signal trap system. The predicted signal peptide sequence with two additional amino acids (1–19) of VdRTX1 protein were fused in‐frame to the mature yeast invertase, enabling secretion of invertase and resulting in yeast growth on YPRAA medium. CMD−W (minus tryptophan, Trp) plates were used to select yeast strain YTK12 transformed with the pSUC2 vector. Growth on YPRAA was used to indicate invertase secretion. The functional signal peptide of Avr1b was used as a positive control. (b) The silencing efficiency of the immunity receptor genes NbBAK1 and NbSOBIR1 on N. benthamiana. N. benthamiana leaves from 3‐week‐old plants were subjected to virus‐induced gene silencing (VIGS) by inoculation with tobacco rattle virus (TRV) constructs TRV:BAK1, TRV:SOBIR1, and TRV:GFP (green fluorescent protein) as a negative control. The silencing efficiency of NbBAK1 or NbSOBIR1 was determined by TRV:BAK1 or TRV:SOBIR1 compared with TRV:GFP using reverse transcription‐quantitative PCR (RT‐qPCR) analysis with N. benthamiana EF‐1α as a reference gene. Means and standard errors from three biological replicates are shown. Asterisks ** indicate significant differences (p < 0.01). (c–e) Analysis of the associations of BAK1 and SOBIR1 with VdRTX1. VdRTX1 was transiently expressed in gene‐silenced plants that were subjected to VIGS by inoculation with TRV constructs for 3 weeks: TRV:BAK1 (c), TRV:SOBIR1 (d), and TRV:GFP (e). Transiently expression of BAX and GFP in the gene‐silenced leaves were used as positive and negative controls, respectively. The phenotypes of induced cell death were photographed 5 days later. (f) Immunoblot analysis of VdRTX1 proteins transiently expressed in gene‐silenced (NbBAK1 or NbSOBIR1) N. benthamiana leaves 5 days following agroinfiltration. Ponceau S‐stained RuBisCO protein was used as a total protein loading control. (g) Cell‐death‐inducing activity analyses using a construct expressing the VdRTX1 protein without the signal peptide (VdRTX1SP). Close to the right of the schematic is a photograph of the N. benthamiana leaves transiently expressing VdRTX1SP protein. The right‐most line indicates immunoblotting results of expressed proteins, in which the top asterisk (*) stands for immunoblotted protein and the bottom hash (#) indicates Ponceau S‐stained RuBisCO protein used as a total protein loading control
FIGURE 4
FIGURE 4
Cell death in Nicotiana benthamiana depends on the nuclear localization of VdRTX1. (a) Identification the cell‐death‐inducing activity of VdRTX1 protein from Verticillium dahliae without the nuclear localization sequence (VdRTX1∆NLS). Close to the right of the schematic is a photograph of the N. benthamiana leaves transiently expressing VdRTX1∆NLS. The right‐most line indicates immunoblotting results of expressed proteins, in which the top asterisk (*) stands for immunoblotted protein and the bottom hash (#) indicates Ponceau S‐stained RuBisCO protein used as a total protein loading control. (b) Subcellular localization of green fluorescent protein (GFP)‐tagged VdRTX1 variants in N. benthamiana. VdRTX1 and VdRTX1∆NLS, N‐terminally tagged in its coding region with a nuclear export signal (VdRTX1NES) and with a nonfunctional mutated NES sequence (VdRTX1mNES) fused with the GFP, were transiently expressed in N. benthamiana leaves. The fluorescent signal of GFP was observed using confocal microscopy. GFP served as a negative control. 4′,6‐diamidino‐2‐phenylindole (DAPI) staining was used to visualize the nuclei. Arrowheads point to the nuclei. Bars, 5 µm. (c) Identification of the cell‐death‐inducing activity of transient expression of VdRTX1NES and VdRTX1mNES in N. benthamiana leaves. The wild‐type VdRTX1 and GFP were used as positive and negative controls, respectively. The phenotype of cell death was photographed at 5 days following agroinfiltration. (d) Immunoblot analyses to confirm protein expression in N. benthamiana leaves transiently expressing the indicated proteins using anti‐HA antibodies. RuBisCO stained by Ponceau S was used as a total protein loading control
FIGURE 5
FIGURE 5
The putative ligand‐binding sites of VdRTX1 are required for cell death induction in Nicotiana benthamiana leaves. (a) Prediction of the putative ligand‐binding sites in VdRTX1 from Verticillium dahliae. The putative ligand‐binding sites were predicted by the online software of I‐TASSER tool (http://zhanglab.ccmb.med.umich.edu/I‐TASSER) and the probability of ligand‐binding sites was filtered with C‐score >0.5. (b) Multiple sequence alignment of typical fungal ribotoxins and nontoxic RNases with the sequence of the VdRTX1 protein. Moderately and highly conserved amino acid residues are shaded grey and black, respectively. The triangles represent the putative ligand‐binding sites, with blue and red representing the divergent and absolutely conserved putative ligand‐binding sites of VdRTX1 compared to known RNases, respectively. (c) Mutation schematic of putative ligand‐binding sites in VdRTX1 for transient expression in N. benthamiana. The putative ligand‐binding sites were individually mutated to an alanine residue. (d) Cell‐death‐inducing activity analyses of the putative ligand‐binding site mutations in VdRTX1. N. benthamiana leaves from 4‐week‐old plants were examined 5 days after transient expression of VdRTX1 mutation constructs. The construct expressing the full‐length VdRTX1 protein‐coding region was used as the positive control. Immunoblotting results of expressed proteins are shown under the leaves, in which the asterisk (*) stands for respective immunoblotted proteins and the hash (#) indicates Ponceau S‐stained RuBisCO protein used as a total protein loading control
FIGURE 6
FIGURE 6
The effector VdCBM1 from Verticillium dahliae suppresses the VdRTX1‐activated plant cell death and defence responses. (a) VdRTX1 from V. dahliae was transiently expressed in 4‐week‐old Nicotiana benthamiana leaves together with VdCBM1 or with VdISC1 by coagroinfiltration of the respective expression constructs. Expression of green fluorescent protein (GFP) with VdRTX1 or VdRTX1 alone were used as cell death references. Photographs depicting cell death lesions were taken at 48 h postinfiltration. (b) Immunoblotting analysis of VdRTX1 and coagroinfiltrated proteins expressed in N. benthamiana leaves. VdRTX1 and VdCBM1 were fused with a FLAG‐tag while VdISC1 and GFP were fused with an HA‐tag. Ponceau S‐stained RuBisCO is shown as a total protein loading control. (c) Electrolyte leakage in N. benthamiana leaves expressing VdRTX1 together with VdCBM1 or the negative control GFP was assayed at 48 h after coagroinfiltration. (d) Callose deposition in N. benthamiana leaves transiently expressing VdRTX1 together with VdCBM1 and the GFP negative control was measured by staining with aniline blue. Bar = 200 µm. (e) Accumulation of reactive oxygen species (ROS) in N. benthamiana leaves was determined at 12 h after coinfiltration of constructs expressing VdRTX1 with VdCBM1 proteins. Leaves were stained with 3,3′‐diaminobenzidine (DAB). (f) Quantitative analysis of DAB staining results of tobacco leaves by ImageJ. Data represent the mean density of DAB‐stained area from three leaves. Statistical significance was calculated using Student's t test. Asterisks *** indicate significant difference (p < 0.001)
FIGURE 7
FIGURE 7
Virulence assays of VdRTX1 deletion mutants on Nicotiana benthamiana and cotton plants. (a) Disease symptoms of N. benthamiana plants inoculated with the various Verticillium dahliae strains. Plants inoculated with sterile water (Mock) and wild‐type (WT) V. dahliae VD8 served as negative and positive controls, respectively. Development of Verticillium wilt symptoms on plants inoculated with the VdRTX1 deletion mutant (ΔVdRTX1) and the corresponding complemented transformant strains (ECVdRTX1‐1 and ECVdRTX1‐2) was observed and photographs were taken 3 weeks following inoculation. There were three replicates of nine plants. (b) Susceptible cotton (Gossypium hirsutum ‘Junmian 1’) seedlings were root‐dipped with sterile water (Mock), wild‐type (WT) V. dahliae VD8, and the VdRTX1 gene deletion mutant (ΔVdRTX1), along with the corresponding complemented transformant strains (ECVdRTX1‐1 and ECVdRTX1‐2). Photographs of disease symptoms were taken 3 weeks following inoculation (top). V. dahliae colonization‐induced plant stem discolouration in longitudinal sections is shown (bottom). There were three replicates of 25 plants each. In planta fungal biomass was assessed by quantitative PCR in N. benthamiana (c) and cotton (d) inoculated with the indicated V. dahliae strains. Error bars represent standard errors. Asterisks ** indicate significant differences (p < 0.01), calculated by unpaired Student's t tests
FIGURE 8
FIGURE 8
Identification and functional analysis of VdRTX1 homologs in Verticillium spp. and other fungi. (a) Identity matrix of VdRTX1 homologs from fungi. Reciprocal BLAST analysis of the VdRTX1 homologs was performed using the BLASTP program (e‐value < 1e−5) to find all pairwise matches among 1162 VdRTX1 homologs. The identity matrix was constructed with all pairwise combinations. The number in parentheses by each fungal grouping represents the total number of VdRTX1 homologs. The blue blocks at the bottom of the identity matrix represent the selected genus for identity comparison with VdRTX1 homologs from Verticillium spp. in (b). Numbers on the left side of the identity matrix represent the 24 selected VdRTX1 homologs examined for cell death detection by transient expression in (d). (b) Identity comparison of VdRTX1 homologs from Verticillium spp. with the selected genus. The left and right numbers in the square brackets represent the total number of VdRTX1 homologs and the number of species in the selected genus, respectively. (c) Sequence alignment of the VdRTX1 homologs from Verticillium spp. (d) Detection of cell death induction by selected VdRTX1 homologs, as indicated on the left side of the identity matrix in (a). Nicotiana benthamiana leaves from 4‐week‐old plants were examined at 5 days following transient expression of the selected homologs. VdRTX1 and green fluorescent protein (GFP) were used as positive and negative controls, respectively
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