Non-host Plant Resistance against Phytophthora capsici Is Mediated in Part by Members of the I2 R Gene Family in Nicotiana spp

Abstract

The identification of host genes associated with resistance to Phytophthora capsici is crucial to developing strategies of control against this oomycete pathogen. Since there are few sources of resistance to P. capsici in crop plants, non-host plants represent a promising source of resistance genes as well as excellent models to study P. capsici - plant interactions. We have previously shown that non-host resistance to P. capsici in Nicotiana spp. is mediated by the recognition of a specific P. capsici effector protein, PcAvr3a1 in a manner that suggests the involvement of a cognate disease resistance (R) genes. Here, we have used virus-induced gene silencing (VIGS) and transgenic tobacco plants expressing dsRNA in Nicotiana spp. to identify candidate R genes that mediate non-host resistance to P. capsici. Silencing of members of the I2 multigene family in the partially resistant plant N. edwardsonii and in the resistant N. tabacum resulted in compromised resistance to P. capsici. VIGS of two other components required for R gene-mediated resistance, EDS1 and SGT1, also enhanced susceptibility to P. capsici in N. edwardsonii, as well as in the susceptible plants N. benthamiana and N. clevelandii. The silencing of I2 family members in N. tabacum also compromised the recognition of PcAvr3a1. These results indicate that in this case, non-host resistance is mediated by the same components normally associated with race-specific resistance.

Keywords: Nicotiana; Phytophthora capsici; R gene; hypersensitive response; non-host resistance.

FIGURE 1

Efficiency of TRV-based VIGS in N. edwardsonii. Three-week-old N. edwardsonii and N. benthamiana plants were agro-infiltrated with Agrobacterium carrying a fragment of the N. benthamiana Sulfur (magnesium chelatase) gene cloned in TRV00 along with Agrobacterium carrying pBintra6 (RNA I) (right). Photographs were taken 4 weeks post-infiltration.

FIGURE 2

Virus-induced gene silencing of EDS1 and I2 enhances susceptibility to P. capsici in N. edwardsonii. Three-week-old plants were infiltrated with Agrobacterium carrying pTRV2-EDS1 or pTRV2-I2 along with Agrobacterium carrying TRV1. At 3 weeks post-infiltration plants were challenged with 5 × 10⁵ zoospores of P. capsici inoculated in the soil near the base of the plant. (A) Top panel are agro-infiltrated plants right before inoculation with P. capsici. Bottom panel are the plants inoculated with P. capsici at 10 dpi. Control non-infiltrated plants (a), agro-infiltrated with pTRV2-Gus (b), pTRV2-EDS1 (c) or pTRV2-I2 (d). Experiments were repeated three times with similar results and representative pictures are presented. (B) Quantification of P. capsici infection in VIGSed N. edwardsonii plants. Controls were non-infiltrated plants or infiltrated with pTRV2-Gus, pTRV2-RanGp2, and PVX-based vector pGR107. Symptoms were monitored at 10 and 14 dpi according to the scale of infection as follows: 0 = Healthy; 1 = 1–20% of the plant affected, Slightly Diseased; 2 = 21–80% of the plant affected, Heavily Diseased; 3 = 81–100% of the plant affected, Dead. N indicate the total number of plants tested from three independent experiments.

FIGURE 3

Resistance to P. capsici is compromised in transgenic tobacco leaves expressing a RNAi construct to silence I2 genes. Infection assay with P. capsici in transgenic tobacco lines expressing a hairpin of I2 was carried out in detached leaves of 4-week-old plants. Representative leaves from wild-type (WT) and different transgenic lines (L20, L8, L5, L9, L12, L2, and L17) inoculated with P. capsici zoospores are shown. Photographs were taken at 3 dpi.

FIGURE 4

Virus-induced gene silencing of SGT1 enhances susceptibility to P. capsici in N. benthamiana and N. clevelandii. Three-week-old N. benthamiana (A,B) and N. clevelandii (C,D) plants were subjected to TRV VIGS using TV-SGT1 (B,D) or pTV00 (A,C). At 3 weeks post-infiltration plants were challenged with 2 × 10⁵ zoospores of P. capsici inoculated in the soil near the base of the plant. Symptoms were monitored at 3, 6, and 9 dpi. Top panels are VIGSed plants right before inoculation with P. capsici. Bottom panels are the plants inoculated with P. capsici at 3 dpi. (E) Disease index (DI = [Σ(number of plants × category value)/total number of plants]) of the SGT1-silenced P. capsici-inoculated N. benthamiana plants at 3, 6, and 9 dpi. A representative experiment is presented from three independent replicates.

FIGURE 5

PcAvr3a1-triggered HR in N. tabacum is compromised in transgenic I2-silenced plants. Leaves from 5- to 6-week-old N. tabacum (cv. Samsun NN) plants were infiltrated with Agrobacterium carrying either empty pBin61 (EV), or constructs expressing PcAvr3a1 or the TMV p50 protein, as indicated. Photographs of representative leaves of WT (Left) and I2-hairpin transgenic (Right) plants were taken at 5 dpi.