Tomato protein kinase 1b mediates signaling of plant responses to necrotrophic fungi and insect herbivory

Abstract

The tomato protein kinase 1 (TPK1b) gene encodes a receptor-like cytoplasmic kinase localized to the plasma membrane. Pathogen infection, mechanical wounding, and oxidative stress induce expression of TPK1b, and reducing TPK1b gene expression through RNA interference (RNAi) increases tomato susceptibility to the necrotrophic fungus Botrytis cinerea and to feeding by larvae of tobacco hornworm (Manduca sexta) but not to the bacterial pathogen Pseudomonas syringae. TPK1b RNAi seedlings are also impaired in ethylene (ET) responses. Notably, susceptibility to Botrytis and insect feeding is correlated with reduced expression of the proteinase inhibitor II gene in response to Botrytis and 1-aminocyclopropane-1-carboxylic acid, the natural precursor of ET, but wild-type expression in response to mechanical wounding and methyl-jasmonate. TPK1b functions independent of JA biosynthesis and response genes required for resistance to Botrytis. TPK1b is a functional kinase with autophosphorylation and Myelin Basis Protein phosphorylation activities. Three residues in the activation segment play a critical role in the kinase activity and in vivo signaling function of TPK1b. In sum, our findings establish a signaling role for TPK1b in an ET-mediated shared defense mechanism for resistance to necrotrophic fungi and herbivorous insects.

Figure 1.

Basal and Induced Expression of the TPK1b Gene. RNA gel blot analysis showing upregulation of TPK1b gene expression by (A) B. cinerea. (B) P. syringae. (C) Mechanical wounding. (D) Oxidative stress (paraquat, methyl viologen). (E) Basal expression in fruit and leaf tissues. Total RNA (15 μg) was loaded per lane. rRNA staining is shown as a loading control. The experiments were repeated at least three times with similar results. Wild-type plants of the tomato cultivar CastlemartII were inoculated with pathogens or were treated with chemicals as described in Methods. h, h after inoculation or chemical treatment; L, leaf; GF, green fruit; GFR, green–red fruit; RF, red fruit; S, stem.

Figure 2.

TPK1b Is a Single-Copy Gene in the Tomato Genome Encoding a Receptor-Like Protein Kinase. (A) DNA gel blot showing the presence of a single copy of TPK1b in the tomato genome. (B) Sequence alignment between TPK1b and related protein kinases. (C) Phylogenetic relationship between TPK1b and related protein kinases. In (A), tomato genomic DNA was digested with EcoRI (EI), Xba I (X), and EcoRV (EV), resolved on agarose gel, transferred to membrane for hybridization, and probed with 5′ end of the TPK1b gene lacking the kinase domain. In (B), the kinase subdomains are marked with a bar underlined and numbered (I to XI). Residues that are conserved in all TPK1b-related kinases are shaded in black, and amino acids that are shared between TPK1b and the other entries are shaded in gray. Sequences were aligned using ClustalW (Thompson et al., 1994). Numbers above the alignment correspond to amino acid positions in TPK1b. The alignment used for the phylogentic tree in (C) is provided as Supplemental Data Set 1 online. Sequences and phylogenic data are from tomato (TPK1b, GenBank accession number EU555286) CAO21648 (Vitis vinifera), MLPK (Brassica rapa), PTO (Lycopersicum esculentum), and Arabidopsis (BIK1/At2g39660, APK1b/At2g28930, APK1a/At1g07570, BIK1-like/At3g55450, APK2b/At2g02800, NAK/At5g02290, and PBS1).

Figure 3.

TPK1b Is Required for Resistance to Botrytis. (A) RT-PCR from wild-type and TPK1b RNAi lines showing TPK1b transcript levels. (B) TPK1b RNAi plants show increased susceptibility to Botrytis. (C) Disease lesion size in Botrytis-inoculated leaves at 2 DAI. (D) Accumulation of Botrytis ActinA mRNA as a measure of fungal growth in drop-inoculated leaves. In (A), numbers above the lanes indicate independent RNAi transgenic lines, and the RNA used for the RT-PCR was extracted from Botrytis-inoculated leaves. In (C), the values represent the mean ± se from a minimum of 60 lesions. Analysis of variance and Duncan's multiple range test were performed to determine the statistical significance of the differences in the mean disease lesion sizes using SAS software (SAS Institute, 1999). Bars with different letters are significantly different from each other at P = 0.05. In (D), RT-PCR was performed using the Botrytis ActinA gene-specific primers. Experiments were repeated at least three times with similar results. h, h after inoculation; d, days after inoculation; BcActin, B. cinerea ActinA gene.

Figure 4.

TPK1b RNAi Plants Show Reduced Resistance to Tobacco Hornworm. (A) Leaf weight recovered at the end of 5 d of feeding trial on detached leaves (n = 25). (B) Larval weight recovered at the end of 5 d of feeding trial on detached leaves (n = 10). (C) Wild-type and TPK1b RNAi plants at the beginning (left) and end (right) of tobacco hornworm feeding trial on whole plants. (D) Size of larvae recovered at the end of tobacco hornworm feeding trial on whole plants. The feeding trails on detached leaf or whole plants were each repeated three times with similar results. In each experiment of detached leaf and whole-plant assays, six and 10 newly hatched larvae (∼9 to 11 mg each), respectively, were placed on at least five 8-week-old plants of each genotype. Larvae were allowed to feed on the same plant for the duration of the trial. In (A) and (B), the data represent the mean ± se from three different experiments. Bars with different letters are significantly different from each other (P = 0.05). Analysis of variance and Duncan's multiple range test were performed as described in the legend for Figure 3.

Figure 5.

Tomato JA and Wound Response Genes Are Required for Botrytis Resistance. (A) Botrytis disease symptoms on leaves of indicated genotypes. (B) Disease lesion size. (C) Fungal growth. (D) Botrytis-induced TPK1b gene expression. (E) Wound-induced TPK1b gene expression in the tomato JA and/or wound response mutants. The pictures in (A) and lesion sizes in (B) are from 3 DAI with Botrytis (3 × 10⁵ spores/mL). Fungal growth in (C) is based on the accumulation of Botrytis ActinA RNA as detected by RNA gel blot hybridization. In (C), total RNA (15 μg) was loaded per lane. In (D) and (E), RT-PCR was performed as described in Methods. The tomato translation initiation factor (eIF4A) gene was amplified in parallel to demonstrate relative quality and quantity of the cDNA. Experiments were repeated at least three times with similar results. h, h after inoculation.

Figure 6.

ET Responses of TPK1b RNAi and Wild-Type Tomato Seedlings. (A) The triple response in the wild type but its absence in TPK1b RNAi seedlings grown in the dark in the presence of increasing concentrations of ACC. (B) Hypocotyl (top) and root (bottom) length of wild-type (black) and TPK1b RNAi (white) seedlings grown in the dark at varying concentrations of ACC. The pictures in (A) and the measurements in (B) were taken at 6 d after plating. Pictures are representative of seedlings of the corresponding treatments. These experiments were repeated at least five times. The asterisk indicates that the data are statistically significant from the corresponding wild-type control (P = 0.05).

Figure 7.

Expression of Tomato Defense-Related Genes in Wild-Type and TPK1b RNAi Tomato Plants. (A) and (B) RT-PCR showing the expression of tomato PI-II during Botrytis infection or mechanical wounding (A) and ACC or MeJA treatment (B). (C) and (D) RNA gel blot showing the expression of tomato ACC synthase and PR-1 during Botrytis infection (C) and mechanical wounding (D). (E) Expression of allene oxide synthase2 during Botrytis infection or mechanical wounding. In (C) to (E), total RNA (15 μg) was loaded per lane, and rRNA is shown as a loading control. Experiments were repeated at least three times with similar results. h, h after Botrytis inoculation or wounding treatment; ACS, ACC synthase; AOS2, allene oxide synthase2.

Figure 8.

Ectopic Expression of Tomato TPK1b Suppresses the Arabidopsis bik1 Mutant Phenotypes and Confers Increased Resistance to Botrytis. (A) RNA blot showing expression of 35S:TPK1b in bik1 and wild-type plants. (B) Botrytis resistance 3 d after spray-inoculation (top panel) and 3 and 5 d after drop inoculation (bottom panels). (C) Accumulation of the Botrytis ActinA mRNA in spray-inoculated plants. In (A) and (C), total RNA (15 μg) was loaded per lane, and rRNA is shown as a loading control. Experiments were repeated at least three times with similar results. h, h after inoculation; d, days after inoculation.

Figure 9.

TPK1b Is a Functional Protein Kinase That Localizes to the Plasma Membrane. (A) Alignment of the activation segment of TPK1b and related kinases. (B) Autophosphorylation and MBP phosphorylation activities of GST-TPK1b and its mutant derivatives in vitro. Top panel, autoradiogram of the gel; bottom panel, Coomassie blue staining. (C) RNA blot showing transgenic expression of TPK1b substitution mutants in the Arabidopsis bik1 mutant. (D) Responses of bik1 plants expressing TPK1b and its substitution mutants to A. brassicicola inoculation. (E) Summary of kinase activity, Botrytis response, and growth-related phenotypes of the Arabidopsis bik1 expressing TPK1b wild type and substitution mutants. In (A), TPK1b activation segment phosphorylatable residues are in red and underlined. Residues that are phosphorylatable and conserved in TPK1b-related kinases are shaded green. In (D), A. brassicicola disease symptoms are from 4 DAI. In (E), the asterisk indicates partial complementation; +, rescues phenotypes or shows auto (TPK1b) or MBP phosphorylation activity; −, fails to rescue the bik1 growth phenotype or shows no TPK1b or MBP phosphorylation activity.

Figure 10.

The N-Myristoylation Motif Is Not Required for TPK1b Subcellular Localization but Is Required for Its Disease Resistance Function. (A) Subcellular localization of the GFP control, TPK1b-GFP, and TPK1b^G2A-GFP fusion proteins in N. benthamiana cells. (B) RNA blot showing transgenic expression of TPK1b-GFP and TPK1b^G2A-GFP. (C) Resistance of Arabidopsis plants expressing TPK1b and TPK1b^G2A to A. brassicicola. The experiments in (A) and (C) were repeated at least three times with similar results.

Figure 11.

TPK1b Is Required for Normal Fruit Structure and Seed Set. (A) TPK1b RNAi plants show defects in fruit structure. (B) TPK1b RNAi plants produce fewer seeds. (C) A comparison of number of seeds/fruit between the wild type and TPK1b RNAi. Representative fruit tissue and seeds from wild-type and TPK1b RNAi plants are shown. In (B), the picture shows part of the fruit and the entire amount of seed recovered from the whole fruit of the corresponding genotype. At least 10 fruits were examined per plant.