Type I J-domain NbMIP1 proteins are required for both Tobacco mosaic virus infection and plant innate immunity

Abstract

Tm-2² is a coiled coil-nucleotide binding-leucine rich repeat resistance protein that confers durable extreme resistance against Tomato mosaic virus (ToMV) and Tobacco mosaic virus (TMV) by recognizing the viral movement protein (MP). Here we report that the Nicotiana benthamiana J-domain MIP1 proteins (NbMIP1s) associate with tobamovirus MP, Tm-2² and SGT1. Silencing of NbMIP1s reduced TMV movement and compromised Tm-2²-mediated resistance against TMV and ToMV. Furthermore, silencing of NbMIP1s reduced the steady-state protein levels of ToMV MP and Tm-2². Moreover, NbMIP1s are required for plant resistance induced by other R genes and the nonhost pathogen Pseudomonas syringae pv. tomato (Pst) DC3000. In addition, we found that SGT1 associates with Tm-2² and is required for Tm-2²-mediated resistance against TMV. These results suggest that NbMIP1s function as co-chaperones during virus infection and plant immunity.

Figure 1. NbMIP1.1a interacts with ToMV MP and Tm-2² in yeast and in vitro.

(A–D) NbMIP1.1a interacts with ToMV MP, LRR domain of Tm-2² (Tm-2²-LRR) and full length Tm-2² (Tm-2²-FL) in yeast. (A, C) Yeast cells harboring BD-ToMV MP (A), BD-Tm-2²-LRR or BD-Tm-2²-FL (C) transformed with AD-NbMIP1.1a grew on Leu^- selection plates and turned blue on X-gal plate containing Gal/Raf but not on plates containing glucose. Yeast cells transformed with either BD or AD alone vector showed no growth on Leu⁻ selection plates and remained white on X-gal plates containing either Gal/Raf or glucose. For each experiment, yeast strains were maintained at 28°C for 5 days. (B, D) Quantification of β-galactosidase activities in yeast two-hybrid interactions. (E, F) GST pull-down assay for detection of in vitro interaction of NbMIP1.1a with ToMV MP (E) or Tm-2² LRR domain (F). For GST pull-down, GST-NbMIP1.1a or GST immobilized on glutathione-Sepharose beads was incubated with gradient dilutions of E. coli-expressed recombinant ToMV MP-3×Flag-6×His protein (E) and Tm-2²-LRR-3×Flag-6×His protein (F) at 1, 10⁻¹, 10⁻². Beads were washed and proteins were analyzed by SDS-PAGE and western blot assays using anti-Flag antibodies.

Figure 2. NbMIP1.1a interacts with ToMV MP and Tm-2² in vivo.

Firefly luciferase complementation imaging assays for in vivo interaction of NbMIP1.1a with ToMV MP (A) and Tm-2² (B). Panels show luminescence images of N. benthamiana leaves agro-infiltrated with nLUC-NbMIP1.1a and ToMV MP-cLUC or Tm-2²-cLUC. The combinations of nLUC-NbMIP1.1a and cLUC, nLUC and ToMV MP-cLUC, nLUC and Tm-2²-cLUC were included as negative controls. (C) NbMIP1.1a co-immunoprecipitated (co-IP) with ToMV MP and Tm-2². NbMIP1.1a-Myc was co-expressed with ToMV MP-HA or Tm-2²-HA in N. benthamiana leaves by agroinfiltration. NbMIP1.1a-Myc co-expressed with HA-nLUC was introduced as a negative control. At 48 hours post infiltration (hpi), leaf lysates were immunoprecipitated with anti-HA beads, then the immunoprecipitates were assessed by western blotting using anti-Myc (upper panel) and anti-HA antibodies (middle panel). In addition to immunoblotting for co-IP, presence of NbMIP1.1a-Myc, ToMV MP-HA, Tm-2²-HA and HA-nLUC in the cell lysates were also analyzed (lower panel).

Figure 3. Tm-2² (VAALLA) mutant, NbMIP1.1a and ToMV MP could exist in the same complex in plants.

(A) The Tm-2² (VAALLA) mutant did not induce HR when co-expressed with ToMV MP-Myc (upper), and failed to induce resistance against TMV-GFP (lower). Tm-2²-HA or Tm-2² (VAALLA)-HA were agroinfiltrated into wild-type N. benthamiana leaves with either ToMV MP-Myc (upper) or TMV-GFP (lower). Photos were taken at 4 dpi under UV light. (B) Tm-2² (VAALLA)-HA co-immunoprecipitated with both ToMV MP and NbMIP1.1a (top). YFP-NbMIP1.1a co-immunoprecipitated with both ToMV MP and Tm-2² (VAALLA) mutant (middle). Loading controls were also analyzed by western blot (bottom). Tm-2² (VAALLA)-HA and YFP-NbMIP1.1a were co-expressed with ToMV MP-Myc or empty Myc vector in N. benthamiana leaves through agroinfiltration. At 60 hpi, leaf lysates were immunoprecipitated with anti-HA or anti-GFP beads, then the immunoprecipitates were assessed by western blotting using anti-HA, anti-Myc or anti-GFP antibodies as indicated.

Figure 4. The subcellular localization of NbMIP1.1a in N. benthamiana cells.

(A) Confocal image of the subcellular localization of NbMIP1.1a in leaf epidermal cells. YFP-NbMIP1.1a was transiently expressed in leaves of N. benthamiana via agroinfiltration and imaged at 48 hpi using a Zeiss LSM 710 laser scanning microscope. YFP signal revealed that NbMIP1.1a is present in the cell membrane, cytoplasm and nucleus. PCD3-1002: a CFP-tagged plasma membrane marker . DAPI: staining for nuclei. Scale bar represents 20 µm. (B) YFP-NbMIP1.1a was found in both the soluble fraction and the membrane fraction (upper panel). Protein extracts were centrifuged at 100,000×g to produce crude soluble (S100) and microsomal (P100) fractions. Fractions were analyzed by western blot following separation by SDS-PAGE. The gels were probed using anti-GFP, anti-V-H-ATPase (vacuolar H-ATPase subunit, a vacuolar membrane marker) and anti-PEPC (phosphoenolpyruvate carboxylase, a cytosolic marker) antibodies as indicated.

Figure 5. Tm-2²-mediated resistance against TMV requires NbMIP1s.

(A) Phenotype of NbMIP1s-silenced and TRV control N. benthamiana plants. NbMIP1s-silenced N. benthamiana plants developed dwarfed stems and crinkled leaves compared to TRV-infected control plants. (B) Leaves of NbMIP1-silenced and TRV control N. benthamiana plants. The leaf edge of NbMIP1s-silenced plants curled downward but TRV-infected control leaves looked normal (right side). Photos were taken at 14 days post agroinfiltration for VIGS. (C) Real time RT-PCR to confirm the suppression of NbMIP1s, and the Actin mRNA levels were used as internal controls. NbMIP1 VIGS: silencing using pTRV2-NbMIP1. (D) Silencing of NbMIP1s caused the appearance of TMV-GFP infection foci and visible HR lesions in the inoculated leaves of NbMIP1s-silenced Tm-2²-containing TM#1 plants. (E) Silencing of NbMIP1s compromised Tm-2²-mediated resistance against TMV, and TMV-GFP spread from the inoculated leaves into the upper non-inoculated leaves of NbMIP1s-silenced TM#1 plants. TRV-infected TM#1 plants were used as negative controls. Photos were taken at 10 days post TMV-GFP infection (dpi). Scale bars represent 1 cm. (F) RT-PCR was performed to confirm the presence of TMV-GFP in systemic leaves of NbMIP1s-silenced TM#1 plants.

Figure 6. NbMIP1s are involved in TMV movement.

(A) Silencing of NbMIP1s reduced cell-to-cell movement of TMV. TMV-GFP formed much smaller infection foci in NbMIP1s-silenced plants (right) compared to control plants (left). Photos were taken at 3 dpi. Scale bars represent 1 cm. (B) Average sizes of TMV-GFP foci at 3 dpi are shown. All values in bar graphs represent means with standard deviation. **: p<0.01 (Student's t-test). Data are from 3 independent experiments, and 9 leaves for each construct per experiment. (C) RT-PCR to confirm that the suppression of NbMIP1s reduced TMV-GFP vRNA levels in local inoculated leaves at 3 dpi. (D) Silencing of NbMIP1s delayed systemic TMV movement. At 5 dpi, TMV-GFP had already spread into the upper non-inoculated leaves in control plants (left), but not into the systemic leaves in NbMIP1s-silenced plants (right). Scale bars represent 1 cm. (E) RT-PCR to confirm the delay of TMV-GFP systemic movement in NbMIP1s-silenced plants at 5 dpi. For each RT-PCR, Actin was used as an internal control.

Figure 7. NbMIP1s are essential for the stability of ToMV MP and Tm-2² protein.

Effect of silencing of NbMIP1s on the steady-state levels of YFP alone (A), ToMV MP-YFP (B) and Tm-2²-YFP (C). Silencing of NbMIP1s reduced the fluorescence intensity of ToMV MP-YFP (B, left) and Tm-2²-YFP (C, left) but not that of YFP alone (A, left). YFP, ToMV MP-YFP and Tm-2²-YFP T-DNA constructs were agroinfiltrated into leaves of NbMIP1s silenced plants and TRV control plants respectively and confocal images (left panels) were taken at 72 hpi. Scale bars represent 20 µm. Proteins were extracted from the agro-infiltrated leaf areas and analyzed by SDS-PAGE, followed by western blot with anti-GFP antibody (right panels). Western blot assays further showed that silencing of NbMIP1s specifically reduced the protein level of ToMV MP-YFP (B, right) and Tm-2²-YFP (C, right) but had no effect on YFP alone (A, right). Ponceau Red staining of Rubisco indicates equal loading (lower panel). All experiments were performed three times with three replicated samples in each experiment.

Figure 8. NbSGT1 interacts with NbMIP1.1a and Tm-2² and is required for Tm-2²-mediated resistance to TMV.

(A–D) NbSGT1 interacts with both NbMIP1.1a and Tm-2² in yeast. (A, C) Yeast cells harboring AD-NbMIP1.1a (A), AD-Tm-2² (C) transformed with AD-NbSGT1 grew on Leu^- selection medium and turned blue on X-gal medium plus Gal/Raf but not on medium plus glucose. Yeast cells transformed with either BD or AD vector alone for control assays showed no growth on Leu⁻ selection medium and remained white on X-gal medium containing either Gal/Raf or glucose. For each experiment, yeast strains were maintained at 28°C for 5 days. (B, D) Quantification of β-galactosidase activities in yeast two-hybrid interactions. (E) NbSGT1 co-immunoprecipitated (co-IP) with NbMIP1.1a and Tm-2². NbSGT1-HA was co-expressed with NbMIP1.1a-Myc or Tm-2²-Myc respectively in N. benthamiana leaves through agroinfiltration. Coexpression of NbSGT1-HA and cLUC-Myc was used as a negative control. At 2 dpi, leaf lysates were immunoprecipitated with anti-HA beads, then the immunoprecipitates were assessed by western blotting using anti-Myc (upper panel) and anti-HA antibodies (middle panel). In addition to immunoblotting for co-IP, presence of NbSGT1-HA, NbMIP1.1a-Myc, Tm-2²-Myc and cLUC-Myc in the cell lysates were also analyzed (lower panel). * indicates nonspecific bands. (F) Silencing of NbSGT1 in Tm-2² transgenic TM#1 plants caused TMV-GFP spreading into the upper non-inoculated leaves (left). TRV-infected TM#1 plants were used as negative controls. Photos were taken at 10 dpi. RT-PCR was performed to confirm the presence of TMV-GFP in systemic leaves of NbSGT1-silenced TM#1 plants (right). (G) Silencing of NbSGT1 reduced the protein level of Tm-2²-Myc. Ponceau Red staining of Rubisco indicates equal loading (lower panel). Experiments were performed three times with three replicated samples in each experiment. (H) Real-time RT-PCR showed that silencing of NbSGT1 had no effect on the expression level of Tm-2²-Myc, and Actin mRNA levels were used as the internal control. Data are shown as means ± SD for 3 independent triplicate experiments (Student's t-test).

Figure 9. NbMIP1s are required for plant immunity mediated by multiple R genes and general elicitors.

(A) Silencing of NbMIP1s resulted in larger TMV-GFP infection foci in transgenic N-containing N. benthamiana plants (NN) plants. Photos were taken under normal or UV light at 7 dpi. (B) Silencing of NbMIP1s compromised N-mediated resistance to TMV-GFP in NN plants and TMV-GFP spread into the upper non-inoculated leaves at 14 dpi. (C) RT-PCR to confirm the presence of TMV-GFP RNA in the upper, non-inoculated leaves of NbMIP1s-silenced NN plants. (D) Silencing of NbMIP1s delayed HR mediated by several R genes and nonhost Pseudomonas syringae pv. tomato DC3000 (Pst DC3000). All R-Avr pairs were expressed in wild type plants. Transient co-expression of Pto with avrPto, or Cf9 with avr9 was performed using agroinfiltration and Pst DC3000 was inoculated in NbMIP1s-silenced and non-silenced control plants. The leaf images of Pst DC3000 were taken at 12 hpi, and the other images corresponding to R-Avr pairs were taken at 3 dpi. Scale bars represent 1 cm. (E) Silencing of NbMIP1s compromised nonhost resistance against Pst DC3000 and caused more bacterial growth. Bacterial growth was monitored at 0, 1, 2 and 3 dpi. Each data point represents the mean ± SEM of 3 replicate samples (** p<0.01, Student's t-test). All experiments were performed at least three times using three or more plants in each experiment.