Signals involved in Arabidopsis resistance to Trichoplusia ni caterpillars induced by virulent and avirulent strains of the phytopathogen Pseudomonas syringae

Abstract

Plants have evolved different but interconnected strategies to defend themselves against herbivorous insects and microbial pathogens. We used an Arabidopsis/Pseudomonas syringae pathosystem to investigate the impact of pathogen-induced defense responses on cabbage looper (Trichoplusia ni) larval feeding. Arabidopsis mutants [npr1, pad4, eds5, and sid2(eds16)] or transgenic plants (nahG) that are more susceptible to microbial pathogens and are compromised in salicylic acid (SA)-dependent defense responses exhibited reduced levels of feeding by T. ni compared with wild-type plants. Consistent with these results, Arabidopsis mutants that are more resistant to microbial pathogens and have elevated levels of SA (cpr1 and cpr6) exhibited enhanced levels of T. ni feeding. These experiments suggested an inverse relationship between an active SA defense pathway and insect feeding. In contrast to these results, there was increased resistance to T. ni in wild-type Arabidopsis ecotype Columbia plants that were infected with P. syringae pv. maculicola strain ES4326 (Psm ES4326) expressing the avirulence genes avrRpt2 or avrB, which elicit a hypersensitive response, high levels of SA accumulation, and systemic acquired resistance to bacterial infection. Similar results were obtained with other ecotypes, including Landsberg erecta, Cape Verdi Islands, and Shakdara. When infected with Psm ES4326(avrRpt2) or Psm ES4326(avrB), nahG transgenic and npr1 mutant plants (which are more susceptible to virulent and avirulent P. syringae strains) failed to show the increased insect resistance exhibited by wild-type plants. It was surprising that wild-type plants, as well as nahG and npr1 plants, infected with Psm ES4326 not expressing avrRpt2 or avrB, which elicits disease, became more susceptible to T. ni. Our results suggest two potentially novel systemic signaling pathways: a systemic response elicited by HR that leads to enhanced T. ni resistance and overrides the SA-mediated increase in T. ni susceptibility, and a SA-independent systemic response induced by virulent pathogens that leads to enhanced susceptibility to T. ni.

Figure 1

Larval weight gain of T. ni feeding for 5 d on Arabidopsis wild-type plants and defense-related mutants. For each experiment, weight gain data were normalized to the weight gain of larvae feeding on wild-type Columbia (Col) plants. The bars represent the least square means (±ses, ANOVA) of relative larval weight gain from four independent experiments. Open bars and hatched bars correspond to the Col and Landsberg erecta (Ler) accessions, respectively. Relative SA levels refer to SA accumulation following infection with Psm ES4326(avrRpt2) or Erysiphe orontii in the case of the npr1, pad4, eds5, eds15, sid2(eds16), and eds1 mutants (Delaney et al., 1995; Zhou et al., 1998; Nawrath and Metraux, 1999; Dewdney et al., 2000; Feys et al., 2001), or the levels in uninfected plants in the case of the acd2, cpr1, and cpr6 mutants (Bowling et al., 1994; Greenberg et al., 1994; Clarke et al., 1998). Relative SA levels: −4, <0.25; −3, 0.25 to 0.5; −2, 0.5 to 0.75; −1, 0.75 to 1; 0, 1.0; +1, 1 to 1.5; +2, 1.5 to 2; +3, 2 to 3; +4, >3-fold wild-type (Wt) levels. The numbers above the bars represent P values adjusted using the Bonferroni method from multiple comparisons between Col wild-type and mutant plants. The P value for Ler shows the difference between Ler and Col wild-type plants. The P values for nahG-Ler and eds1-2 are for the comparisons with Ler wild-type plants. ns, Not significant; P > 0.05, * 0.01 < P < 0.05, **P < 0.01.

Figure 2

Relative defoliation rate of T. ni feeding on various Arabidopsis wild-type plants and defense-related mutants. The bars represent the means (±ses) of the relative defoliation rates obtained by comparing the extent of defoliation for each particular ecotype, transgenic, or mutant plant with the average extent of defoliation observed for the relevant wild-type plants that had been planted side by side with the particular experimental plants. The asterisks indicate the significance level, determined by permutation tests, of the defoliation differences between control plants (Col in the case of nahG, npr1, pad4, eds5, sid2, acd2, cpr1, cpr6, and Ler indicated by open bars, and Ler in the case of nahG-Ler and eds1-2 indicated by hatched bars) and experimental plants. ns, Not significant; P > 0.05, * 0.01< P < 0.05, ** P < 0.01.

Figure 3

Scatterplot of relative defoliation rates against relative larval weight gains, showing a strong positive correlation between larval weight gains and defoliation rates (r² = 0.92, P < 0.001).

Figure 4

Larval weight gain of T. ni feeding on various Arabidopsis ecotypes infiltrated with isogenic virulent and avirulent strains of Psm ES4326. As described in “Materials and Methods,” lower leaves were inoculated with 10 mm MgSO₄ or with Psm ES4326(pLAFR3), Psm ES4326(avrRpt2), or Psm ES4326(avrB). Four days later, the inoculated leaves were removed and newly hatched T. ni larvae were placed on the upper leaves. Larval weight gain was measured after 5 d of feeding. For each experiment, weight gain data were normalized to the weight gain of larvae feeding on wild-type Col plants inoculated with 10 mm MgSO₄. The bars represent the least square means (±ses, ANOVA) of the relative larval weight gain data from three independent experiments. The numbers above each bar correspond to the Bonferroni adjusted P values from multiple t tests. For each ecotype, weight gain of T. ni larvae feeding on bacterial infected plants were compared with that on MgSO₄-treated plants of the same ecotype. ns, Not significant; P > 0.05, * 0.01 < P < 0.05, ** P < 0.01.

Figure 5

Disease symptoms that developed on wild-type ecotype Col plants or on rps2 or rpm1 mutant plants 4 d after they were inoculated with Psm ES4326(pLAFR3), Psm ES4326(avrRpt2), or Psm ES4326(avrB).

Figure 6

Larval weight gain of T. ni feeding on wild-type Arabidopsis plants and R gene mutant plants that were infiltrated with various strains of Psm ES4326. As described in “Materials and Methods,” lower leaves were inoculated with 10 mm MgSO₄ or with Psm ES4326(pLAFR), Psm ES4326(avrRpt2), or Psm ES4326(avrB). Four days later, the inoculated leaves were removed and newly hatched T. ni larvae were placed on the upper leaves. Larval weight gain was measured after 5 d of feeding. For each experiment, weight gain data were normalized to the weight gain of larvae feeding on mock-inoculated wild-type Col plants. The bars represent the means (±ses, ANOVA) of relative weight gain from four independent experiments. The numbers above each bar correspond to the Bonferroni adjusted P values from multiple t tests. The P values for bacteria-treated wild-type or R gene mutant plants are from multiple comparisons against mock-treated wild-type or R gene mutant plants, correspondingly. ns, Not significant; P > 0.05, * 0.01 < P < 0.05, ** P < 0.01.

Figure 7

Larval weight gain of T. ni feeding on Arabidopsis defense-related mutants infiltrated with various strains of Psm ES4326. As described in “Materials and Methods,” lower leaves were inoculated with 10 mm MgSO₄ or with Psm ES4326(pLAFR), Psm ES4326(avrRpt2), or Psm ES4326(avrB). Four days later, the inoculated leaves were removed and newly hatched T. ni larvae were placed on the upper leaves. Larval weight gain was measured after 5 d of feeding. For each experiment, weight gain data were normalized to the weight gain of larvae feeding on mock-inoculated wild-type Col plants. The bars represent the means (±ses, ANOVA) of relative weight gain from four independent experiments. The numbers above each bar correspond to the Bonferroni adjusted P values from multiple t tests. The P values for bacteria-treated wild-type or defense-related mutant plants are from the multiple comparisons against mock-treated wild-type or defense-related mutant plants, respectively. ns, Not significant; P > 0.05, * 0.01 < P < 0.05, ** P < 0.01.

Figure 8

Model of the signaling leading to insect resistance or sensitivity after pathogen infection. Infection with an avirulent pathogen increases SA levels, which has been shown to cause insect sensitivity. However, the simplest interpretation of the data presented in this paper is that an unknown signal from an avirulent infection apparently overrides the SA signaling, thereby increasing insect resistance. This signal may partly depend on HR and SA. Infection with a virulent pathogen appears to result in another unidentified signal that systemically increases insect sensitivity in an SA-independent manner.