Mechanisms Of Helicoverpa Zea Salivary ATP Hydrolyzing Enzymes In Mediating Defenses In Tomato Plant

The larva of Helicoverpa zea (Lepidoptera:Noctuidae) is a major agricultural pest. They are mainly found in North America except for northern Canada and Alaska and can cause extensive economic loss on a wide variety of crops, such as corn, rice, wheat, cotton, soybeans, and so on. Tomato, Solanum lycopersicum, an annual herb of the family Solanaceae is a very important vegetable crop worldwide. Present world production of tomato is more than one hundred and fifty million tones. However, losses due to pests are estimated to be about34.4%of attainable tomato yield under current production practices. Common tomato pests in the United States include H. zea, Halyomorpha picus, Manduca quinquemaculata, and other insect species. Current researches on H. zea are mainly focus on its morphology, biology, ecology and chemical control aspects, while little was known about the relationships between H. zea and their host plants. The saliva and oral secretions of herbivores have been reported to be important cues in mediating inducible plant responses in the last two decades. In order to elucidate the relationships between herbivores and their host plants, researchers are engaged in identifying and separating those effectors from herbivores' saliva and oral secretion. Supported by the USDA AFRI program (2010-65105-20639). H. zea was chosen with one of its hosts, tomato, was investigated in our study to explore how H. zea saliva, especially ATP hydrolyzing enzymes, mediate tomato plant defenses. The major results are summarized as follows:1Detection of ATP hydrolysis activity in different tissues and saliva of H. zeaBased on the importance of extracellular ATP (eATP) in plant physiological responses, H. zea saliva was collected for ATP hydrolysis activity possibility test. Five microliters of plant eATP was added to cuvette and reacted with5μl H. zea saliva for5min. We detected93.9±3.4nmol-min-1·mg-1ATP hydrolysis specific activity in0.2±0.0μg·μl-1H. zea saliva, and it degraded0.4±0.0nmol·L-1eATP from tomato leaves.The results of in-gel ATP hydrolysis activity test showed that ATP hydrolyzing enzymes are ubiquitous among different tissues of H. zea. Although the same amount of total crude homogenate (50μg) was loaded on the gels, the activity stained bands were much stronger in labial glands than in extracts from the other tissues.2Gene cloning and expression of three ATP hydrolysis-related genes in H. zea 2.1Gene cloning of three ATP hydrolysis-related genes in H. zeaTo further investigate the function of each ATP hydrolyzing enzyme in mediating plant defenses, the cDNAs of H. zea labial gland apyrase, ATP synthase and ATPase13A1were cloned. The sequencing results showed that their complete cDNAs consisted of2,102,1,789and4,171bp respectively, with open reading frames of1,641,1,551and3,483nucleotides, respectively. They encode proteins of546,516and1160amino acids residues, and their calculated theoretical molecular masses are61.2,55.2and130.2kDa, respectively. The sequences have been deposited in GenBank (accession numbers:HM569605, HM156739and HQ184468).Besides, the presence of N-terminal signal peptide was predicted in three amino acid sequences, since it was proved to be functional in translocating proteins across membranes to extracellular space, which indicate the potential secretion of these three ATP hydrolyzing enzymes from the labial glands to the saliva. The results showed that no signal peptide was found in the ATP synthase and ATPase13A1proteins, but one was present in apyrase. Additionally, ATPase13A1and ATP synthase were tested as non-classically secreted proteins, with NN-scores of0.767and0.492respectively. An NN-score above0.5indicates a high probability of the protein being secreted.2.2Expression levels of three ATP hydrolysis-related genes in different tissues of H. zeaThe comparative qRT-PCR revealed that the relative expression of ATP synthase and ATPase13A1genes was significant higher than they were in other tissues (P<0.05). The relative expression level of apyrase was highest in Malpighian tubules, which was1.5-fold higher than it was in labial glands. The results demonstrated that ATP synthase and ATPase13A1play important role in the salivary glands, and apyrase might play a role in the excretory and osmoregulatory systems in H. zea.Because the synthesis and secretion of salivary proteins are diet and host dependent, relative expression levels of ATPase13A1gene in labial glands of H. zea fed different diets were tested to see the interaction between plant defenses and saliva gene expression. The results showed that expression of ATPase13A1gene could be suppressed by methyl jasmonate induced plants, but not by feeding on the JA-deficient def1plants or when3μg·g-1jasmonic acid was added to diets. These results suggest that some component(s), other than jasmonic acid, in induced plants inhibits ATPase13A1expression.3Expression of the ATP hydrolyzing enzymes in a heterologous system The in vitro expressions of H. zea apyrase, ATP synthase and ATPase13A1were performed successfully in the pET system. The results of expression time-courses analysis of the fusion proteins suggested that the protein yields of H. zea apyrase and ATP synthase were continual higher when they were induced with1.0mmol·L-1IPTG for longer time, even overnight. However, the reproduction ability of cells containing pET-43.1b(+)-ATPase13A1recombinant plasmid would be inhibited after five hours induction with1.0mmol·L-1IPTG, which indicated that the existence of certain concentration of target ATPase13A1had effect on cell growth and accordingly, the expression level of ATPase13A1fusion protein decreased.4Protein purification and activity testSDS-PAGE analysis of the purified proteins indicated that the molecular masses were60.0,54.5and51.7kDa for apyrase, ATP synthase and ATPase13A1, respectively.Apyrase has been verified to be present in H. zea saliva, salivary glands and purified protein by western blot analysis.The native-PAGE analysis and ATP hydrolysis activity test results both showed that the target purified proteins were activated after the last step of purification. The specific activities were196.5,219.8and83.7nmol·min·mg-1, with111.9,231.5and70.4%yield for apyrase, ATP synthase and ATPase13A1, respectively.5Plant responses to purified ATP hydrolyzing enzymes from H. zeaPurified apyrase, ATP synthase and ATPase13A1with a similar total ATP hydrolysis activity of74.3pmol·L-1·min-1were each applied separately to wound site on the tomato leaf. The results demonstrated that all the seven genes were induced significantly after wounding (P<0.05). However, treatments with different protein concentrations of purified apyrase, ATP synthase and ATPase13A1at the same total ATP hydrolysis activity after wounding suppressed the expressions of PIN2, ARG, PPO and TD genes significantly (P<0.05), which indicated that the ATP hydrolyzing enzymes from H. zea inhibited the tomato jasmonic acid pathway significantly (P<0.05), especially at higher specific activities. As far as the protein concentrations of purified protein are concerned, the higher protein concentrations with lower specific activities weakened their effects on the jasmonic acid pathway in wounded tomato leaves, and accordingly, restored this defense response of tomato leaves to wounding.Besides, the results of qRT-PCR showed that PAL and PR-10genes, which both are associated with the salicylic acid pathway, had reversed reactions to these enzymes, that is, expression of PAL was suppressed by lower specific activities of apyrase and ATPase13A1, but by higher specific activities of ATP synthase. As for PR-10gene levels, they were totally different. The difference between the gene responses to ATP synthase and to other two proteins might be caused by the dual function of both hydrolysis and synthase activity in ATP synthase. Because of the limitations in our method of testing for ATP hydrolysis activity, it could not be determined if there was any ATP synthesized during measurement. More research about the ATP synthase activity of this protein is required. The relative expression levels of PR-10genes revealed that higher specific activities of ATP synthase and the lowest specific activities of ATPase13A1induced the salicylic acid pathway significantly (P<0.05).Besides, the suppressed OSM gene expression levels by higher specific activities of these proteins suggest that these three ATP hydrolyzing enzymes possibly mediate plant defenses through inhibiting the ethylene pathway.6Glandular trichome production assaysGlandular trichomes have been known as delayed induced plant defenses against herbivores for over five decades. The glandular trichome production was studied after10days treatment with the highest concentration of each purified protein. The results demonstrated that wounding did induce the trichome productions in new tomato leaves, and the density of glandular trichomes were significantly higher than in unwounded plants (P<0.05). However, after treatments with purified ATP hydrolyzing enzymes, there was no significant difference between the trichome numbers in wounded and unwounded plants (P>0.05). This indicated that ATP hydrolyzing enzyme treatments suppressed the effects of wounding on new leaves in tomato plants. These results are consistent with the qRT-PCR analysis results of plant defensive genes, since the jasmonic acid pathway has been verified to be the key channel that regulates the development of glandular trichomes in tomato.7Role of H. zea apyrase in suppression host defensesOur most intriguing finding is that herbivorous insects may employ a similar mechanism to limit host eATP accumulation and signaling. This is strikingly similar to one of the mechanisms used by blood-feeding arthropods to overcome host defenses, namely the production of ATP-hydrolyzing enzymes such as apyrase that degrade eATP. Mosquito salivary apyrases, as is the H. zea apyrase, are members of the5'-nucleotidase family of apyrases. Hence the comparatively high degree of sequence similarity of the H. zea salivary apyrase with mosquito apyrases suggests a much broader evolutionary role for salivary apyrases than previously envisioned.