Arabidopsis NDR1 is an integrin-like protein with a role in fluid loss and plasma membrane-cell wall adhesion - PubMed (original) (raw)
Arabidopsis NDR1 is an integrin-like protein with a role in fluid loss and plasma membrane-cell wall adhesion
Caleb Knepper et al. Plant Physiol. 2011 May.
Abstract
Arabidopsis (Arabidopsis thaliana) NON-RACE-SPECIFIC DISEASE RESISTANCE1 (NDR1), a plasma membrane-localized protein, plays an essential role in resistance mediated by the coiled-coil-nucleotide-binding site-leucine-rich repeat class of resistance (R) proteins, which includes RESISTANCE TO PSEUDOMONAS SYRINGAE2 (RPS2), RESISTANCE TO PSEUDOMONAS SYRINGAE PV MACULICOLA1, and RPS5. Infection with Pseudomonas syringae pv tomato DC3000 expressing the bacterial effector proteins AvrRpt2, AvrB, and AvrPphB activates resistance by the aforementioned R proteins. Whereas the genetic requirement for NDR1 in plant disease resistance signaling has been detailed, our study focuses on determining a global, physiological role for NDR1. Through the use of homology modeling and structure threading, NDR1 was predicted to have a high degree of structural similarity to Arabidopsis LATE EMBRYOGENESIS ABUNDANT14, a protein implicated in abiotic stress responses. Specific protein motifs also point to a degree of homology with mammalian integrins, well-characterized proteins involved in adhesion and signaling. This structural homology led us to examine a physiological role for NDR1 in preventing fluid loss and maintaining cell integrity through plasma membrane-cell wall adhesions. Our results show a substantial alteration in induced (i.e. pathogen-inoculated) electrolyte leakage and a compromised pathogen-associated molecular pattern-triggered immune response in ndr1-1 mutant plants. As an extension of these analyses, using a combination of genetic and cell biology-based approaches, we have identified a role for NDR1 in mediating plasma membrane-cell wall adhesions. Taken together, our data point to a broad role for NDR1 both in mediating primary cellular functions in Arabidopsis through maintaining the integrity of the cell wall-plasma membrane connection and as a key signaling component of these responses during pathogen infection.
Figures
Figure 1.
Homology modeling of NDR1 with the integrin-like protein LEA14. A, Predicted structure of NDR1. B, The solved structure of LEA14 (pdb_1yyc; Singh et al., 2005). C, NDR1 predicted structure threaded onto the solved structure of LEA14, highlighting the predicted structural homology. N denotes N terminal. PHYRE analysis returned an estimated structural homology precision of 95%, with an E-value of 0.008. Additional predicted models are shown in
Supplemental Figure S1
.
Figure 2.
A to D, Enhanced electrolyte leakage in the ndr1-1 mutant following inoculation with Pst DC3000. Levels of electrolyte leakage from Col-0 and ndr1-1 plants in response to DC3000 inoculation are displayed as percentage maximal leakage. Treatments include Pst DC3000 expressing vector control (A), AvrRpt2 (B), AvrB (C), and AvrPphB (D). The dramatic increase in leakage observed in ndr1-1 as compared with Col-0 when inoculated with Pst DC3000 correlates with the onset of disease symptoms. dpi, Days postinoculation. Error bars display
sd
from four technical replicates from two to three biological replicates. Significance was determined using two-way ANOVA, where asterisks represent statistically significant differences between Col-0 and ndr1-1: * P < 0.05, *** P < 0.001. E, Col-0 and ndr1-1 leaves at 0, 2, 3, and 4 d postinoculation with Pst DC3000 expressing AvrRpt2.
Figure 3.
Relative mRNA expression of biotic and abiotic responsive genes in ndr1-1 following Pst DC3000 inoculation. A, Altered levels of expression in wild-type Col-0 and ndr1-1 mutant plants of LEA family genes in response to Pst DC3000 or Pst DC3000 expressing AvrRpt2, AvrB, or AvrPphB. B, Expression levels of aquaporin homologs known to be drought responsive in Arabidopsis when inoculated with Pst DC3000 or Pst DC3000 expressing AvrRpt2. Error bars display
sd
from one to two technical replicates from two biological replicates. Samples were taken at 0, 24, and 48 hpi. Expression is displayed as 0-h average fold for Col-0. Significance was determined using two-way ANOVA, where asterisks represent statistically significant differences between Col-0 and ndr1-1 and pound signs represent statistically significant changes over time: *,# P < 0.05; **,## P < 0.01; ***,### P < 0.001.
Figure 4.
NDR1 associates with itself and RIN4 in planta, and this association is unaffected by mutations in the NGD site. Agrobacterium expressing HA:NDR1 or HA:RIN4, along with Agrobacterium expressing T7:NDR1 with substitutions in the NGD motif (wild type, NGD, RGD, or AAA), was infiltrated into N. benthamiana. Tagged proteins were immunoprecipitated from samples taken 48 h after infiltration using anti-HA (NDR1 or RIN4; right lane) or anti-T7 (NDR1; left lane) antibody. Proteins were detected by blotting with anti-T7 (top blot) or anti-HA (bottom blot) HRP-conjugated antibody. A, T7:NDR1 NGD-HA:NDR1. B, T7:NDR1 AAA-HA:NDR1. C, T7:NDR1 RGD-HA:NDR1. D, T7:NDR1 NGD-HARIN4. E, T7:NDR1 AAA-HA:RIN4. F, T7:NDR1 RGD-HA:RIN4. Western blots in
Supplemental Figure S6
show relative detection limits.
Figure 5.
ndr1-1 exhibits altered PAMP responses. A and B, The expression levels of FRK1 mRNA was analyzed by qRT-PCR in Col-0 and ndr1-1 mutant plants in response to flg22 or mock treatment. Error bars display
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from two technical replicates from one biological replicate. Expression is displayed as untreated average fold for Col-0. Statistical significance was determined using one-way ANOVA followed by Tukey’s test, where asterisks represent statistically significant differences as compared with untreated Col-0 and pound signs represents statistically significant differences between Col-0 and ndr1-1: * P < 0.05; **,## P < 0.01; *** P < 0.001. C, Western blot analysis of MAPK3/6 in Col-0 and ndr1-1 mutant plants in response to flg22 or mock treatment.
Figure 6.
Growth of Pst DC3000 in Arabidopsis is altered by mutations to the NGD site of NDR1. Bacterial growth assay of Pst DC3000 (EV) and Pst DC3000 expressing AvrRpt2, AvrB, or AvrPphB dip inoculated on ndr1-1 lines complemented with native NDR1 promoter with NDR1 NGD (ndr1-1/PNDR1:NGD; A), NDR1 RGD (ndr1-1/PNDR1:RGD; B), or NDR1 AAA (ndr1-1/PNDR1:AAA; C). Growth was assayed at 0 and 4 d postinoculation. Growth is expressed as log cfu cm−2. Error bars display
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from three technical replicates from two biological replicates. Statistical significance was determined using unbalanced two-way ANOVA (model included in
Supplemental Fig. S7
), where asterisks represent statistically significant differences between Col-0 and ndr1-1: * P < 0.1, ** P < 0.01, *** P < 0.0001 (for P values, see
Supplemental Table S3
).
Figure 7.
ndr1-1 mutant plants exhibit altered plasmolysis and plasma membrane-cell wall focal adhesions. A, Time course (left to right: 0 [before], 2, and 5 min after treatment) of 8-d-old Col-0 or ndr1-1 CaCl2-plasmolyzed hypocotyls. In contrast to plasmolyzed wild-type Col-0 hypocotyl cells, which reveal plasma membrane-cell wall attachments (arrows), the plasma membrane of ndr1-1 cells quickly loses adhesion following the induction of plasmolysis, yielding spherical protoplasts within the cell wall. Bars = 25 μm. B, Complemented ndr1-1 mutant line constitutively expressing a GFP:NDR1 protein exhibits wild-type plasma membrane-cell wall adhesions (arrows). Differential interference contrast (top panel) and confocal-differential interference contrast overlay (bottom panel) images of CaCl2-induced plasmolysis are shown. Arrows indicate Hechtian strand formation, illustrating significant physical linkages with the plasma membrane and cell wall. Bars = 30 μm. C, Hechtian strands are absent, or significantly reduced, in the ndr1-1 mutant plant. Bar = 10 μm.
Figure 8.
Application of exogenous peptides can alter the plasma membrane-cell wall adhesion in wild-type Col-0. Application of exogenous VNGDG or VRGDG peptide results in reduced plasma membrane-cell wall adhesions in Col-0, ndr1-1/P_NDR1_:NGD, and ndr1-1/P_NDR1_:RGD hypocotyl cells. Both ndr1-1 and ndr1-1/P_NDR1_:AAA hypocotyls are unaltered in adhesion phenotypes upon addition of purified VRGDG, VNGDG, and VAAAG peptides. Arrows indicate cell wall adhesion points. Differential interference contrast microscopy images were collected 5 min after the induction of plasmolysis. Bars = 25 μm.
References
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