Identification of genes required for Cf-dependent hypersensitive cell death by combined proteomic and RNA interfering analyses

Abstract

Identification of hypersensitive cell death (HCD) regulators is essential to dissect the molecular mechanisms underlying plant disease resistance. In this study, combined proteomic and RNA interfering (RNAi) analyses were employed to identify genes required for the HCD conferred by the tomato resistance gene Cf-4 and the Cladosporium fulvum avirulence gene Avr4. Forty-nine proteins differentially expressed in the tomato seedlings mounting and those not mounting Cf-4/Avr4-dependent HCD were identified through proteomic analysis. Among them were a variety of defence-related proteins including a cysteine protease, Pip1, an operative target of another C. fulvum effector, Avr2. Additionally, glutathione-mediated antioxidation is a major response to Cf-4/Avr4-dependent HCD. Functional analysis through tobacco rattle virus-induced gene silencing and transient RNAi assays of the chosen 16 differentially expressed proteins revealed that seven genes, which encode Pip1 homologue NbPip1, a SIPK type MAP kinase Nbf4, an asparagine synthetase NbAsn, a trypsin inhibitor LeMir-like protein NbMir, a small GTP-binding protein, a late embryogenesis-like protein, and an ASR4-like protein, were required for Cf-4/Avr4-dependent HCD. Furthermore, the former four genes were essential for Cf-9/Avr9-dependent HCD; NbPip1, NbAsn, and NbMir, but not Nbf4, affected a nonadaptive bacterial pathogen Xanthomonas oryzae pv. oryzae-induced HCD in Nicotiana benthamiana. These data demonstrate that Pip1 and LeMir may play a general role in HCD and plant immunity and that the application of combined proteomic and RNA interfering analyses is an efficient strategy to identify genes required for HCD, disease resistance, and probably other biological processes in plants.

Fig. 1.

Two-dimensional polyacrylamide gel electrophoresis profiles of proteins from Cf-4/Avr4 tomato seedlings mounting a hypersensitive cell death (HCD). (A) Profile from Cf-4/Avr4 tomato seedlings (HCD⁺). (B) Profile from Cf-4 tomato seedlings (HCD⁻). A total of 300 μg protein per gel was loaded. After 2-D PAGE using IPG strips (24 cm, pH 3–7 NL), the gels were silver stained. Protein spots that expressed differentially over two-fold in the cotyledons of the HCD⁺ and HCD⁻ seedlings are numbered and indicated with arrows in the gels in which the spots displayed with higher abundance. Spot numbers are as given in Supplementary Table S3.

Fig. 2.

Close-up view of the protein spots differentially expressed between Cf-4/Avr4 (hypersensitive cell death; HCD⁺) and Cf-4 (HCD⁻) tomato seedlings. The differentially expressed protein spots are circled and grouped according to their functions.

Fig. 3.

Functional classification of the identified proteins differentially expressed in Cf-4/Avr4 (hypersensitive cell death; HCD⁺) and Cf-4 (HCD⁻) tomato seedlings.

Fig. 4.

Influence of virus-induced gene silencing (VIGS)-inducing treatment for six genes on growth and development of N. benthamiana plants. Genes subjected to VIGS analyses encoded (A–C) a proteasome 20S beta 1.1 subunit (NbPb, 19), (D) a luminal-binding protein (NbBiP, 58), (E, F) a SIPK type MAP kinase (Nbf4, b12), (G, H) an asparagine synthetase (NbAsn, b48), (I) an ASR (Abscissic acid, Stress, Ripening) protein (NbASR, b67), and (J) a chloroplast thiazole biosynthetic protein (NbTHI, b73). Plants were infiltrated with cell suspensions of Agrobacterium transformed with TRV VIGS vector carrying a fragment of the target gene or just empty vector (EV; K, L). The photographs were taken 12 days (A, B), 14 days (D, K), 19 days (C), 21 days (G, J), 30 days (I, L), and 42 days (E, F, H) after agroinfiltration. Spot numbers at bottom right are as given in Supplementary Table S3.

Fig. 5.

Effect of virus-induced gene silencing (VIGS)-inducing treatment for five genes on Cf-4/Avr4-dependent hypersensitive cell death in N. benthamiana plants. (A) Empty vector control (EV). Genes subjected to VIGS analyses encode (B) a Phytophthora-inhibited protease 1 (Pip1)-like protein (59), (C) a SIPK type of MAP kinase (b12), (D) an asparagine synthetase (b48), (E) a LeMir-like protein (b60), and (F) a subtilisin-like protease (b44). Plants were infiltrated with cell suspensions of Agrobacterium transformed with TRV VIGS vector carrying a fragment of the target gene or just empty vector (EV). The photographs were taken 3 days after agroinfiltration. Spot numbers at bottom right are as given in Supplementary Table S3.

Fig. 6.

RT-PCR analysis for confirmation of the gene silencing in virus-induced gene silencing (VIGS)-treated plants. Mesophyll tissues of leaf areas corresponding to the place for agroinfiltration for induction of Cf-4/Avr4-dependent hypersensitive cell death in either a target gene-VIGS-treated N. benthamiana plants (VIGS) or an empty vector-VIGS-treated plants (CK1) were sampled for total RNA isolation 3 weeks after VIGS-inducing agroinfiltration. Products from 30 cycles of RT-PCR (CK2: without RTase) were loaded. Actin was used as an inner standard gene for loading check. Spot numbers are as given in Supplementary Table S3.

Fig. 7.

Virus-induced gene silencing analysis for function of four genes in Cf-4/Avr4-dependent hypersensitive cell death in tomato plants: (A) empty vector control (EV), (B, C) a Phytophthora-inhibited protease 1 (Pip1)-like protein (59), (D) a SIPK type of MAP kinase (b12), (E) an asparagine synthetase (b48), and (F) a LeMir-like protein (b60). The analysis is similar to that for Fig. 5 except that tomato plants were used. Spot numbers at bottom right are as given in Supplementary Table S3.

Fig. 8.

Virus-induced gene silencing analysis for function of four genes in Cf-9/Avr9-dependent hypersensitive cell death (HCD) in N. benthamiana plants: (A) empty vector control (EV), (B) a Phytophthora-inhibited protease 1 (Pip1)-like protein (59), (C) a SIPK type of MAP kinase (b12), (D, E) an asparagine synthetase (b48), and (F) a LeMir-like protein (b60). The analysis is similar to that for Fig. 5 except that Cf-9/Avr9-dependent HCD instead of Cf-4/Avr4-dependent HCD was checked. Spot numbers at bottom right are as given in Supplementary Table S3.

Fig. 9.

Virus-induced gene silencing analysis for function of four genes in X. oryzae pv. oryzae (Xoo)-induced hypersensitive cell death (HCD) in N. benthamiana plants: (A) empty vector control (EV), (B) a Phytophthora-inhibited protease 1 (Pip1)-like protein (59), (C) a SIPK type of MAP kinase (b12), (D, E) an asparagine synthetase (b48), and (F) a LeMir-like protein (b60). The analysis is similar to that for Fig. 5 except that Xoo-induced HCD instead of Cf-4/Avr4-dependent HCD was checked. Spot numbers at bottom right are as given in Supplementary Table S3.

Fig. 10.

RNAi analysis for function of Pip1 in Cf-4/Avr4- and Cf-9/Avr9-dependent hypersensitive cell death (HCD) and X. oryzae pv. oryzae-induced HCD in tobacco plants. A Pip1 RNAi construct pC1305-35S::PIP1-RNAi was made, which carried a hairpin sequence cassette that comprised of two copies of a 300bp fragment of Pip1 flanking the intron sequence of P. vulgaris nitrite reductase gene (U10419) in the opposite direction. Agrobacterium transformed with this construct was infiltrated into all sectors of left half leaves while Agrobacterium transformed with empty vector (EV) as control, into right half leaves of Sumsun NN tobacco plants. Two days after agroinfiltration, HCD in each sector of the RNAi-treated leaves was evaluated as described above.