Plant immune response to pathogens differs with changing temperatures

Abstract

Temperature fluctuation is a key determinant for microbial invasion and host evasion. In contrast to mammals that maintain constant body temperature, plant temperature oscillates on a daily basis. It remains elusive how plants operate inducible defenses in response to temperature fluctuation. Here we report that ambient temperature changes lead to pronounced shifts of the following two distinct plant immune responses: pattern-triggered immunity (PTI) and effector-triggered immunity (ETI). Plants preferentially activate ETI signaling at relatively low temperatures (10-23 °C), whereas they switch to PTI signaling at moderately elevated temperatures (23-32 °C). The Arabidopsis arp6 and hta9hta11 mutants, phenocopying plants grown at elevated temperatures, exhibit enhanced PTI and yet reduced ETI responses. As the secretion of bacterial effectors favours low temperatures, whereas bacteria multiply vigorously at elevated temperatures accompanied with increased microbe-associated molecular pattern production, our findings suggest that temperature oscillation might have driven dynamic co-evolution of distinct plant immune signaling responding to pathogen physiological changes.

Figure 1

Elevated temperatures promote gene activation in PTI signaling. (A) flg22-induced WRKY29 activation in Arabidopsis leaves and protoplasts at different temperatures. Leaves or protoplasts from four-week-old plants were treated with H₂O or 100 nM flg22 for 3 hrs for RNA isolation and real-time RT-PCR (qRT-PCR) analysis. The expression of WRKY29 was normalized to the expression of UBQ10. (B) flg22-induced FRK1 activation in Arabidopsis protoplasts at different temperatures. Protoplasts from four-week-old plants were treated with H₂O or 100 nM flg22 for 3 hrs for RNA isolation and qRT-PCR analysis. The expression of FRK1 was normalized to the expression of UBQ10. The gene activation fold is presented as the ratio of flg22 treatment to H₂O treatment with the mean ± s.e.m. (n=3) from three independent biological replicates. (C) Activation of pWRKY29::LUC by different MAMPs at different temperatures. The protoplasts were transfected with pWRKY29::LUC and pUBQ::GUS as an internal control, and treated with 10 nM flg22, 10 nM HrpZ, 50 μg/ml chitin, or 20 nM NPP1 for 3 hrs at the indicated temperatures. GST is the control for NPP1. The promoter activity was shown as the ratio of relative luciferase activity to GUS activity. * indicates a significant difference with p<0.05 analyzed with SPSS software one-way ANOVA analysis when compared with corresponding data from 16°C. The above experiments were repeated three times with similar results.

Figure 2

Elevated temperatures promote early PTI signaling. (A) flg22-induced MAPK activation at different temperatures. Ten-day old WT seedlings were treated with 100 nM flg22 at different temperatures for indicated time. MAPK activation was detected with an α-pERK antibody and Coomassie Brilliant Blue staining of Rubisco (RBC) protein is shown for equal loading control. (B) flg22-induced BIK1 phosphorylation in protoplasts at different temperatures. The band intensity of pMPK3, pMPK6, BIK1 and pBIK1 was quantified by the Image J software and presented with mean ± s.e.m. (n=3) from three independent biological replicates. * indicates a significant difference with p<0.05 analyzed with SPSS software one-way ANOVA analysis when compared with corresponding data from 16°C. The above experiments were repeated three times with similar results.

Figure 3

Elevated temperatures inhibit ETI responses. (A) Activation of WRKY46 by AvrRpt2 at different temperatures. Four-week-old Dex-AvrRpt2 plants were hand-inoculated with H₂O or 10 μM Dex, or the protoplasts were transfected with AvrRpt2 or a vector control, and incubated at different temperatures for 6 hr before sample collection for RNA isolation. The gene activation fold is presented as the ratio of AvrRpt2 expression to controls with the mean ± s.e.m. (n=3) from three independent biological replicates. (B) Activation of WRKY46 by AvrRpm1 or AvrB at different temperatures. The protoplasts were transfected with AvrRpm1, AvrB or a vector control, and incubated at different temperatures for 6 hr before sample collection for RNA isolation. * indicates a significant difference with p<0.05 analyzed with SPSS software one-way ANOVA analysis when compared with corresponding data from 16°C. (C) Cell death in DEX-avrRpt2 transgenic plants at different temperatures. The DEX-avrRpt2 transgenic plants were hand-inoculated with H₂O or 10 μM Dex, and incubated at different temperatures. The cell death was recorded 24 hpi for plants incubated at 16, 23, 28 and 32°C, 40 hpi for plants at 10°C, and 48 hpi for plants at 4°C. The cell death was shown by Trypan blue staining and % indicates the percentage of wilting leaves of total inoculated leaves. The expression of avrRpt2 after DEX treatment is shown. Actin is the control for RT-PCR. The above experiments were repeated three times with similar results.

Figure 4

Elevated temperatures do not suppress expression of bacterial effector and plant defense-related genes. (A) Expression of effector proteins at different temperatures. The protoplasts were transfected with AvrRpt2, AvrRpm1, or AvrB, and incubated at different temperatures for 6 hr before sample collection for Western blot with an α-HA antibody. Western blot with an α-RBC (Rubisco) antibody is shown as a loading control. (B) RPS2 protein level at 23°C and 32°C. The pRPS2::RPS2-HA plants were incubated at 23°C and 32°C for 9 hr before sample collection for Western blot with an α-HA antibody. (C) Expression of plant resistance and signaling genes at 23°C and 32°C by RT-PCR analysis. The plants were incubated 23°C and 32°C for 6 hr before sample collection for RNA isolation. AvrRpt2-mediated RIN4 degradation (D) and AvrRpm1-mediated RIN4 phosphorylation (E) at different temperatures. Dex-AvrRpt2-HA (in Col-0 background) or Dex-AvrRpm1-HA transgenic plants (in rpm1 background) were infiltrated with 10μM Dex or H₂O for 8 hr. Immunoblot was performed with an α-RIN4 or α-HA antibody. Staining of RBC shows equal loading. The above experiments were repeated three times with similar results.

Figure 5

Enhanced PTI responses in arp6-10 and hta9hta11 mutant plants. (A) flg22-induced MAPK activation in WT and mutants. Ten-day old seedlings were treated with 100 nM flg22 at room temperature for indicated time. MAPK activation was detected with an α-pERK antibody. The band intensity of pMPK3 and pMPK6 was quantified by the Image J software and presented with mean ± s.e.m. (n=3) from three independent biological replicates. * indicates a significant difference with p<0.05 analyzed with SPSS software one-way ANOVA analysis when compared with data from WT plants. (B) flg22-induced PTI marker gene expression. 10-day-old seedlings of WT and mutants were treated with 100 nM flg22 for 0.5 or 1 hr. The gene expression of FRK1 and At2g17740 was detected by qRT-PCR and normalized to the expression of UBQ10. The data are shown as the mean ± s.e.m. from three independent biological replicates. * indicates a significant difference with p<0.05 analyzed with SPSS software one-way ANOVA analysis when compared with data from WT plants. The above experiments were repeated three times with similar results.

Figure 6

Reduced ETI cell death in arp6-10 and hta9hta11 mutant plants. Compromised HR triggered by Pst avrRpt2 (A) and avrRpm1 (B) in arp6-10 and hta9/11 mutant plants. Four-week-old WT and mutant plants were hand-inoculated with bacteria at a concentration of 1 × 10⁸ cfu/ml. HR was examined by counting the percentage of wilting leaves of total inoculated leaves (>20) at different time points after inoculation. Electrolyte leakage induced by Pst avrRpt2 (C) and avrRpm1 (D) was reduced in arp6-10 and hta9/11 mutant plants. Five leaf discs were excised from 4-week-old plants hand-inoculated with bacteria at 1 × 10⁸ cfu/ml for each sample at each time point with three replicates. * indicates a significant difference with p<0.05 analyzed with SPSS software one-way ANOVA analysis when compared with data from WT plants. The above experiments were repeated three times with similar results.

Figure 7

Compromised ETI-mediated restriction of bacterial growth and defense gene activation in arp6-10 and hta9hta11 mutant plants. (A) Bacterial growth assay. Four-week-old plants were hand-inoculated with Pst avrRpm1 or avrRpt2 at 5 × 10⁵ cfu/ml. The bacterial growth was measured 0 days post-inoculation (dpi) or 4 dpi. (B) ETI marker gene expression. Four-week-old plants were hand-inoculated with bacteria at 1 × 10⁷ cfu/ml, and RNA was collected 6 hpi for qRT-PCR analysis. The expression of AIG1 and PR1 was normalized to the expression of UBQ10. The data are shown as the mean ± s.e.m. (n=3) from three independent biological replicates and the asterisk (*) indicates a significant difference with p<0.05 analyzed with SPSS software one-way ANOVA analysis when compared with data from WT plants. (C) A model of temperature operation of distinct plant innate immune responses. At low ambient temperatures, bacteria secrete a large suite of virulence effectors to promote pathogenicity (ETS), which in turn stimulates plants to co-evolve and preferentially activate ETI signaling. At the elevated temperatures, bacteria multiply vigorously and produce increased amount of MAMPs, which stimulate plants to switch to PTI signaling. Ambient temperature fluctuation likely drives the dynamic co-evolution of bacterial pathogenesis and host immunity. The above experiments were repeated three times with similar results.