Non-host Resistance Induced by the Xanthomonas Effector XopQ Is Widespread within the Genus Nicotiana and Functionally Depends on EDS1

Abstract

Most Gram-negative plant pathogenic bacteria translocate effector proteins (T3Es) directly into plant cells via a conserved type III secretion system, which is essential for pathogenicity in susceptible plants. In resistant plants, recognition of some T3Es is mediated by corresponding resistance (R) genes or R proteins and induces effector triggered immunity (ETI) that often results in programmed cell death reactions. The identification of R genes and understanding their evolution/distribution bears great potential for the generation of resistant crop plants. We focus on T3Es from Xanthomonas campestris pv. vesicatoria (Xcv), the causal agent of bacterial spot disease on pepper and tomato plants. Here, 86 Solanaceae lines mainly of the genus Nicotiana were screened for phenotypical reactions after Agrobacterium tumefaciens-mediated transient expression of 21 different Xcv effectors to (i) identify new plant lines for T3E characterization, (ii) analyze conservation/evolution of putative R genes and (iii) identify promising plant lines as repertoire for R gene isolation. The effectors provoked different reactions on closely related plant lines indicative of a high variability and evolution rate of potential R genes. In some cases, putative R genes were conserved within a plant species but not within superordinate phylogenetical units. Interestingly, the effector XopQ was recognized by several Nicotiana spp. lines, and Xcv infection assays revealed that XopQ is a host range determinant in many Nicotiana species. Non-host resistance against Xcv and XopQ recognition in N. benthamiana required EDS1, strongly suggesting the presence of a TIR domain-containing XopQ-specific R protein in these plant lines. XopQ is a conserved effector among most xanthomonads, pointing out the XopQ-recognizing R_xopQ as candidate for targeted crop improvement.

Keywords: EDS1; ETI; Nicotiana benthamiana; Non-host resistance; Solanaceae; Xanthomonas; XopC; XopQ.

Figure 1

Plant phenotypes resulting from Agrobacterium-mediated effector expression. T3Es from Xcv were transiently expressed in 86 Solanaceae lines via Agrobacterium-mediated T-DNA transfer (see Table 2 for details). Plant reactions were classified into five groups, each represented by a color: red, fast cell death (3 dpi); orange, cell death (8 dpi); yellow, chlorosis (8 dpi); orange/yellow striped, chlorosis or cell death (8 dpi); white, no visible reaction (8 dpi). As examples, phenotypes of four plant lines after expression of different T3Es are shown. Plant lines were abbreviated according to Table S2. The Xcv effector causing the respective reaction is indicated. Photographs were taken 8 dpi.

Figure 2

Plant reactions to Agrobacterium-mediated transient expression of Xcv T3Es. Heatmap representation of effector responses in 86 different non-host Solanaceae plant lines (for abbreviations see Table S2). Five plants per line, two leaves per plant, resulting in 10 spots per Agrobacterium strain, were inoculated with Agrobacterium strains mediating expression of the T3Es indicated on top. Plant reactions observed on at least 7/10 spots were classified as follows: fast cell death (3 dpi); cell death (6 dpi); chlorosis (6 dpi); chlorosis or cell death (6 dpi); no visible reaction (6 dpi). Reactions on only 4-6/10 spots were judged to be inconsistent. Plant reactions were visualized in a heatmap using the color code indicated. Each reaction type was assigned a value serving as the basis for clustering. The dendrogram shows the results of hierarchical clustering using average linkage and euklidean distance measures for T3Es and plant genotypes, respectively.

Figure 3

Avirulence activity of XopQ is restricted to Nicotiana species. Eighty-six different non-host Solanaceae plant lines (for abbreviations see Table S2) were inoculated with Xcv strains 85-10 and Xcv 85-10ΔxopQ, harboring empty vector (ev) or pBRM:xopQ (pxopQ), at OD₆₀₀ = 0.4; reactions were scored for 6 days. Five plants per line with two leaves per plant were inoculated resulting in 10 spots per analyzed Xcv strain. Plant reactions are indicated according to the following color code: red, fast cell death (3 dpi); orange, cell death (6 dpi); yellow, chlorosis (6 dpi); orange/yellow striped, chlorosis or cell death (6 dpi); green, water-soaked lesions (6 dpi); white, no visible reaction (6 dpi). Colors were assigned if the same type of reaction was observed on ≥7/10 spots, reactions on only 4-6/10 spots were judged inconsistent, indicated in gray. Plant phenotypes 8 dpi of Agrobacterium mediating xopQ expression are indicated on the right-hand side of each column.

Figure 4

XopQ shows avirulence activity in Nicotiana spp. Five non-host lines were infected with Xcv: N. benthamiana (Nbent), N. tabacum (Ntab), N. paniculata (Npan), N. clevelandii (Ncle), and N. rustica (Nrus). (A) Leaves were inoculated with Xcv strains 85-10, 85-10ΔxopQ, and 85-10ΔhrcN, harboring empty vector (ev) or pBRM:xopQ (pxopQ) at OD₆₀₀ = 0.4. Photographs were taken 6 dpi (Nbent, Ntab), 7 dpi (Npan, Ncle) and 12 dpi (Nrus), respectively. (B,C) Bacterial growth of Xcv strains in leaves was tested. The same Xcv strains as above were inoculated and bacterial multiplication was monitored over a period of 10 days. Values represent the mean of three samples from three different plants. Error bars indicate standard deviation. Different letters represent statistically significant differences; asterisks indicate statistically significant differences when compared to the wild-type strain (two sided t-test, P < 0.05). Experiments were repeated at least twice with similar results.

Figure 5

XopC influences Xcv-mediated non-host resistance in Solanum americanum. Solanum americanum (Same 1) leaves were inoculated with Xcv strains 85-10, 85-10ΔxopC, and 85-10ΔhrcN, harboring an empty vector (ev) or pBRM:xopC (pxopC), at OD₆₀₀ = 0.4. Plant reactions were documented 5 dpi (upper panel) and 3 dpi (lower panel). For better visualization of cell death reactions at 3 dpi, the leaf was bleached in EtOH (lower panel). The experiment was repeated twice with similar results.

Figure 6

Recognition of XopQ and Xcv in N. benthamiana depends on EDS1. (A) Schematic representation of EDS1 loci in N. benthamiana. The genomic mutations harbored in the Nbeds1a-1 line are indicated. (B–E) Leaves of wild-type N. benthamiana (EDS1) and Nbeds1a-1 (eds1) mutant plants were inoculated. (B) A. tumefaciens strains mediating expression of the indicated T3Es and GFP with OD₆₀₀ = 0.8 were inoculated. Photographs were taken 7 dpi. (C) A. tumefaciens strains mediating expression of XopQ, GFP or NbEDS1a with OD₆₀₀ = 0.8 were mixed in a 1:1 ratio and inoculated. Photographs were taken 10 dpi. (D) Inoculation of Xcv 85-10 and 85-10ΔxopQ at OD₆₀₀ = 0.4. Phenotypes were documented 7 dpi. (E) Bacterial multiplication was monitored over a period of 6 days after inoculation of Xcv 85-10 and 85-10ΔxopQ at OD₆₀₀ = 0.0004. Values represent the mean of three samples from three different plants. Error bars indicate standard deviations. Asterisks indicate significant differences compared to Xcv 85-10 in EDS1 plants (two-sided t-test, P < 0.05). Experiments were repeated at least twice with similar results.

Figure 7

N. tabacum recognizes T3Es similarly to its progenitors. T3Es which trigger consistent plant reactions in N. tomentiformis, N. sylvestris and at least 21 of 46 tested N. tabacum lines were compared. For details see Table 3.