Induced ER chaperones regulate a receptor-like kinase to mediate antiviral innate immune response in plants

Abstract

Mounting an effective innate immune response against pathogens requires the rapid and global reprogramming of host cellular processes. Here we employed complementary proteomic methods to identify differentially regulated proteins early during a plant's defense response. Besides defense-related proteins, constituents of the largest category of upregulated proteins were cytoplasmic- and ER-residing molecular chaperones. Investigating the significance of upregulated ER chaperones, we find that silencing of ER-resident protein disulfide isomerases NbERp57 and NbP5 and the calreticulins NbCRT2 and NbCRT3 led to partial loss of N immune receptor-mediated defense against Tobacco mosaic virus (TMV). Furthermore, NbCRT2 and NbCRT3 were required for the expression of a previously uncharacterized induced receptor-like kinase (IRK). IRK is a plasma membrane-localized protein required for N-mediated hypersensitive response, programmed cell death, and resistance to TMV. These data support a model in which ER-resident chaperones are required for the accumulation of membrane-bound or secreted proteins during plant innate immunity.

Figure 1. Schematics of strategy used for coordinated induction of defense response and proteomics method workflow

(A) N. benthamiana plants with and without the N immune receptor were grown at room temperature (RT, ∼22-24°C) for four weeks (Left panel). The plants were shifted to 32°C overnight and TMV was inoculated on all of the leaves and allowed to replicate for 3 days at 32°C (Middle panel). The plants were shifted back to RT and samples were collected at 0, 2, 8, and 16 h post temperature shift (Left panel). (B) The DIGE (left) and the iTRAQ (right) workflow is shown. These workflows converged when samples were analyzed by mass spectrometry to identify differentially regulated proteins.

Figure 2. Representative illustration of differentially regulated proteins from DIGE and iTRAQ analyses

(A) A cropped DIGE gel image. The first dimension is pH via isoelectric focusing and the second dimension is molecular weight via SDS-PAGE. Green spots are down-regulated and orange/red spots are up-regulated. Yellow spots exhibit no change in regulation. The circled numbered spots were identified by mass spectrometry (Table 1). (B-E) Three dimensional volumetric models for DIGE spots were created and measured (circled white) with Decyder MS (B and C) and compared with centroided iTRAQ data displayed with Comet Viewer (D and E) from the time point, T=8h. Panels B and D represent quantification of SGT1 and panels C and E represent the quantification of PR-Q. The right section of DIGE panels (B and C) and the peak at 114.1 in ITRAQ panels (D and E) represent proteins or peptides from plants not undergoing a defense response. The left section of DIGE panels (B and C) and the peak at 117.1 in ITRAQ panels (D and E) represent proteins or peptides from plants undergoing a defense response.

Figure 3. Validation of up-regulated chaperones during defense response

Western blot analyses were conducted with CRT, HSP90 or HSC70 antibodies using protein extracted from 0, 2, 8, and 16 h treated plant tissue without N that did not undergo a defense response and plant tissue with N immune receptor that did undergo a defense response. Protein concentration was normalized using a Bradford assay and coomassie-stained RuBisCo was used as a loading control. The protein ladder was Magic Marker XP (M).

Figure 4. ER chaperones function is required for N immune receptor-mediated resistance to TMV

(A) N-containing N. benthamiana plants were infiltrated with Agrobacterium containing VIGS-Vector control, VIGS-N, VIGS-NbERp57, VIGS-NbP5, VIGS-NbCRT3 and VIGS-NbCRT2. VIGS-silenced plants were infected with TMV-U1 approximately 10 days after the introduction of silencing constructs. Pictures were taken of the whole plant (top row) and the upper leaves (bottom row). HR-PCD spread with the movement of TMV, which was characterized by a collapse of tissue and yellowish-brown death. TMV did not move in the VIGS-Vector control (column 1) and moved strongly in VIGS-N (column 2). Movement was observed in the VIGS-NbERp57, VIGS-NbP5, VIGS-NbCRT3 and NbCRT2 silenced plants (columns 3-6). (B) TMV movement from inoculated leaves into upper uninoculated leaves was verified using semi-quantitative RT-PCR of the TMV movement protein (MP) (top Row). TMV-MP was detected in the VIGS-NbERp57, VIGS-NbP5, VIGS-NbCRT3, and VIGS-NbCRT2 (columns 3-6) plants, but less when compared to VIGS-N (column 2). No TMV-MP was detectable in the VIGS-Vector control (column 1). Levels of EF1α were used as a quantity control (bottom row). (C) Western blot analyses were conducted with CRT antibodies using protein extracted from three biological replicates of VIGS-vector control (lanes 1-3), VIGS-NbCRT3 (lanes 4-6) and VIGS-NbCRT2 (lanes 7-9) silenced plants. Calnexin (top band) is unaffected by the silencing. NbCRT2 (middle band) is greatly reduced in the VIGS-NbCRT2 plants. The NbCRT3 (bottom band) is undetectable in the VIGS-NbCRT3 plants.

Figure 5. Plasma membrane localized IRK function is required for defense response and accumulation is dependent on CRT

(A) The up-regulation of NbIRK was verified by RT-PCR. There is a strong increase in NbIRK expression between 2 – 16 h after the induction of defense (top panel). No RT (C) and an uninfected mock tissue (M) were used as controls. Levels of EF1α were used as a quantity control (bottom panel). (B) Confocal images of NbIRK-Citrine (left column) or AtIRK-Citrine (right column) co-expressed with RIN4-Cerulean. NbIRK-Citrine and AtIRK-Citrine are co-localized with RIN4-Cerulean around the border of the cell in the plasma membrane. Chloroplasts auto fluoresce red. Scale bar represents 20 μm. (C) N-containing N. benthamiana plants were infiltrated with Agrobacterium containing VIGS-Vector control or VIGS-IRK. VIGS-silenced plants were infected with TMV-U1 approximately 10 days after the introduction of silencing constructs. Pictures were taken of the whole plant (top row) and the upper leaves (bottom row). HR-PCD spread with the movement of TMV, which was characterized by a collapse of tissue and yellowish-brown death in the upper uninoculated leaves of VIGS-IRK (right column). TMV did not move in the VIGS-Vector control (left column). (D) Movement of TMV was verified using semi-quantitative RT-PCR of TMV-MP (top panels). TMV-MP was detected in the VIGS-IRK plants (right column) but not in the VIGS-Vector control (left column). Levels of EF1α were used as a quantity control (bottom panels). (E) HR-PCD assay in VIGS-Vector (left column) and VIGS-IRK (right column) silenced N-containing N. benthamiana plants. TMV-p50 was expressed via Agrobacterium transient expression. Two independent replicates show decreased HR in IRK-silenced plants compared to the VIGS-Vector control. (F) IRK-Citrine and RIN4-Citrine were expressed in plants silenced with VIGS-Vector control (lanes 1-3), VIGS-NbCRT3 (lanes 4-6) and VIGS-NbCRT2 (lanes 7-9). Protein was extracted from three independent biological replicates and immunoblot analysis was performed with GFP antibodies. The relative quantity of IRK-Citrine is strongly reduced in VIGS-NbCRT3 and partially reduced in VIGS-NbCRT2. Quantity of RIN4-Citrine was not affected. Protein concentration was normalized using a Bradford assay and coomassie staining of the membrane was used as a loading control.