Different epitopes of Ralstonia solanacearum effector RipAW are recognized by two Nicotiana species and trigger immune responses

Abstract

Diverse pathogen effectors convergently target conserved components in plant immunity guarded by intracellular nucleotide-binding domain leucine-rich repeat receptors (NLRs) and activate effector-triggered immunity (ETI), often causing cell death. Little is known of the differences underlying ETI in different plants triggered by the same effector. In this study, we demonstrated that effector RipAW triggers ETI on Nicotiana benthamiana and Nicotiana tabacum. Both the first 107 amino acids (N_1-107 ) and RipAW E3-ligase activity are required but not sufficient for triggering ETI on N. benthamiana. However, on N. tabacum, the N_1-107 fragment is essential and sufficient for inducing cell death. The first 60 amino acids of the protein are not essential for RipAW-triggered cell death on either N. benthamiana or N. tabacum. Furthermore, simultaneous mutation of both R75 and R78 disrupts RipAW-triggered ETI on N. tabacum, but not on N. benthamiana. In addition, N. tabacum recognizes more RipAW orthologs than N. benthamiana. These data showcase the commonalities and specificities of RipAW-activated ETI in two evolutionally related species, suggesting Nicotiana species have acquired different abilities to perceive RipAW and activate plant defences during plant-pathogen co-evolution.

Keywords: Nicotiana; Ralstonia solanacearum; E3 ligase; RipAW; cell death; effector; effector-triggered immunity.

FIGURE 1

RipAW triggers cell death on Nicotiana benthamiana. Five‐week‐old Nicotiana spp. were leaf‐inoculated with Ralstonia solanacearum strains or agrobacteria containing the effector constructs. (a, b) R. solanacearum strains GMI1000, CFBP2957, and CIP301 trigger cell death in N. benthamiana (a) and Nicotiana tabacum (b). Leaves inoculated with R. solanacearum strains were digitally photographed at 4 days postinoculation (dpi). (c) RipAW, but not RipAU, RipAF1, and RipAZ1, is able to trigger cell death in N. benthamiana. The photographs of leaves expressing those effectors were taken with a camera at 3 dpi. (d) Expression of RipAW enhances accumulation of NbPR1. The total RNA was extracted from the agroinfiltrated N. benthamiana leaves with RipAW and green fluorescent protein (GFP) control at 24 hours postinoculation (hpi). The expression of NbPR‐1 was determined by reverse transcription quantitative PCR and normalized to NbActin. (e) Expression of RipAW limits R. solanacearum JY1 growth. RipAW and control GFP were agroinfiltrated into N. benthamiana leaves. After 24 h, R. solanacearum JY1 strain solution (10⁵ cfu) was infiltrated into the same leaves. Then the JY strain growth in leaves expressing RipAW was measured at the indicated times. Significant differences were evaluated with Student's t test (**p < 0.01). (f) NbSGT1 is required for RipAW‐triggered cell death on N. benthamiana. The first two true leaves of 2‐week‐old N. benthamiana plants were infiltrated with agrobacteria carrying TRV1, TRV2::NbSGT1, TRV2::GFP, and TRV2::NbPDS. Then NbSGT1‐silencing plants were agroinfiltrated with RipAW and investigated for RipAW‐triggered cell death. The picture of RipAW‐triggered cell death on N. benthamiana was taken at 5 dpi. All experiments were repeated three times with similar results. Strain names have been shortened in the image as follows: GMI1000 (GM), CFBP2957 (CF), CIP301 (CP), IPO1609 (IP), UW551 (UW), NCPPB3987 (NC), and UY031 (UY). H represents the number of leaves showing cell death, T represents the total number of R. solanacearum inoculated leaves

FIGURE 2

E3 ligase activity is essential for RipAW‐triggered cell death in Nicotiana benthamiana, but not in Nicotiana tabacum. RipAW and its derivatives were transiently expressed in leaves of 5‐week‐old Nicotiana spp. by agroinfiltration. (a) Schematic illustration of RipAW and its derivatives RipAW (ΔNEL), RipAW (ΔN_1‐107), and RipAW (C177A). (b) Loss of E3 ligase activity abolishes RipAW‐triggered cell death in N. benthamiana. Cell death phenotype on N. benthamiana was photographed at 4 days postinoculation (dpi) with a digital camera. (c) Cell death in (b) was quantified by investigating electrolyte leakage. The N. benthamiana leaves expressing RipAW and its derivatives were harvested at 4 dpi and the conductivity was measured by a conductimeter. Electrolyte leakage was manifested as the percentage of sample conductivity_unboiling/conductivity_boiling (the following assay is similar). Different letters above the columns indicate significant differences between conditions (one‐way analysis of variance, Tukey's test, p < 0.01). (d) E3 ligase activity is not required for RipAW‐triggered cell death in N. tabacum. Photographs of cell death in N. tabacum induced by RipAW and its mutants were taken at 4 dpi. (e) Cell death in (d) was quantified by measuring electrolyte leakage. The agroinfiltrated leaves were collected at 4 dpi and their electrolyte leakage was measured with a conductimeter. Asterisks indicate significant differences between RipAW mutants and RipAW (FL) (Student's t test, ****p < 0.001). (f) Detection of RipAW and its derivatives in N. benthamiana with western blot using anti‐GFP antibody. Leaves expressing RipAW and its mutants were harvested and protein was extracted around 24 h after agroinfiltration. H is the number of leaves with cell death and T is the total number of infiltrated leaves. All experiments were repeated three times with the similar results

FIGURE 3

The first 60 N‐terminal amino acids are not required for RipAW‐triggered cell death on Nicotiana spp. RipAW N‐terminal deletion mutants were agroinfiltrated into leaves of 5‐week‐old N. benthamiana and N. tabacum. (a) Schematic illustration of RipAW N‐terminal mutants. (b) Deletion of the first 60 amino acids does not affect RipAW‐triggered cell death on N. benthamiana. Leaves agroinfiltrated with RipAW mutants were digitally photographed at 4 days postinoculation (dpi). (c) Cell death triggered by the truncated RipAW variants in (b) was quantified by measuring ion leakage. N. benthamiana leaves expressing the truncated RipAW variants were collected at 4 dpi and immersed into double‐deionized water. Then their ion leakage was measured with a conductimeter. Different letters above the columns indicate significant differences between conditions (one‐way analysis of variance, Tukey's test, p < 0.01). (d) The first 60 amino acids are not essential for RipAW‐triggered N. tabacum cell death. N. tabacum leaves agroinfitrated with the truncated RipAW mutants were photographed at 5 dpi. (e) Detection of RipAW and its mutant variants in N. benthamiana with western blot using anti‐FLAG antibody. The leaves expressing RipAW and its mutants were harvested and protein was extracted around 24 h after agroinfiltration. H is the number of leaves displaying cell death and T is the total number of infiltrated leaves. All experiments were repeated three times with similar results

FIGURE 4

RipAW orthologs exhibit differential abilities to trigger cell death on Nicotiana spp. RipAW members from different strains were transiently expressed on leaves of 5‐week‐old Nicotiana spp. by agroinifiltration. (a) RipAW_RS orthologs triggered strong cell death on N. benthamiana. Leaves expressing RipAW orthologs were photographed at 4 days postinoculation (dpi). (b) Cell death in (a) was quantified by measuring electrolyte leakage. The conductivities of N. benthamiana leaves agroinfiltrated with RipAW orthologs were measured with a conductimeter at 4 dpi. Different letters above columns indicate significant differences between conditions (one‐way analysis of variance, Tukey's test, p < 0.01). (c) The expression of RipAW orthologs in N. benthamiana was detected by western blot using anti‐FLAG antibody. Leaves expressing RipAW orthologs were harvested and protein was extracted at 24 h after agroinfiltration. (d) RipAW_RS and RipAW_Po triggered N. tabacum cell death. Leaves agroinfiltrated with RipAW family members were photographed at 5 dpi. H is the number of leaves with cell death and T is the total number of infiltrated leaves. The experiments were repeated three times with similar results

FIGURE 5

RipAW (N_1‐90)_CF and RipAW (N_1‐107)_RS induce strong cell death on Nicotiana tabacum. (a) Expression of RipAW (N_1‐90)_CF and RipAW (N_1‐107)_RS caused strong cell death. Leaves transiently expressing the N‐terminus of RipAW orthologs were photographed at 4 days postinoculation (dpi). (b) Cell death in (a) was quantified by testing electrolyte leakage. Leaves agroinfiltrated with the N‐terminus of RipAW orthologs were collected at 4 dpi and immersed in double‐deionized water. Ion leakage was measured with a conductimeter. Different letters above the columns indicate significant differences between conditions (one‐way analysis of variance, Tukey's test, p < 0.01). H is the number of dead leaves and T is the total number of infiltrated leaves. The experiments were repeated three times with similar results

FIGURE 6

Arginine in positions 75 and 78 of RipAW is essential for cell death in Nicotiana tabacum. The indicated RipAW versions were agroinfiltrated into leaves of 5‐week‐old Nicotiana spp. (a) Substitution of both R75/R78 for alanine and H62/H64 for aspartate did not affect tissue collapse elicited by RipAW in N. benthamiana. Cell death symptoms on leaves expressing RipAW (R75/78A) and RipAW (H62/64D) were photographed at 4 days postinoculation (dpi). (b) Cell death induced by RipAW (R75/78A) and RipAW (H62/64D) on N. benthamiana was quantified by electrolyte leakage. Leaves expressing RipAW (R75/78A) and RipAW (H62/64D) were collected at 4 dpi and immersed in double‐deionized water. The conductivity of samples was measured by a conductimeter. Different letters above columns indicate the significant difference (one‐way analysis of variance, Tukey's test, p < 0.01). (c) Simultaneous mutation on R75 and R78 abolished cell death triggered by RipAW on N. tabacum. Cell death symptoms caused by RipAW (R75/78A) were photographed and the leaves showing cell death were counted at 4 dpi. (d) Cell death in (c) was quantified by electrolyte leakage assay. N. tabacum leaves agroinfiltrated with RipAW and its point mutants were harvested at 4 dpi and put into double‐deionized water. Electrolyte leakage was detected by a conductimeter. Asterisks indicate statistically significant differences between RipAW and its variants (Student's t test, ****p < 0.001). (e) Expression of RipAW and its mutants was detected by western blot with anti‐FLAG antibody in N. benthamiana. Leaves expressing RipAW point mutants were harvested and protein was extracted 24 h after agroinfiltration. H is the number of leaves with cell death and T is the total number of infiltrated leaves. The experiments were repeated three times with the similar results