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6.3.2 Genes differentially expressed during host alternation and subsequent insights Whilst no consistent gene expression changes could be found between aphids present early and late on apple, differences were clear when the aphid moved onto plantain. A problem with the early-late analyses may be any molecular underpinning leading up to host alternation was too subtle to be detected by differentially displayed bands. Also the ‘decision’ to produce winged morphs and host alternate is quite variable, with some colonies already having the odd winged aphid as early as March. The apple-plantain analyses, however, does suggest what might be inducing host alternation. Table 8. Genes differentially expressed by RAA on its primary and secondary hosts
In contrast to the gene functions seen in the resistance/susceptibility profiling, the genes differentially expressed during host alternation tend to be involved in obtaining nourishment. Two genes for amino acid synthesis and one for nitrate/nitrite transport are noteworthy. As the aphid’s diet is phloem sap, which is known to be deficient in amino acids and nutrients, it would appear that RAA needs to upregulate these genes to cope with deficiencies in their alternate hosts. Arabinose efflux permease is involved in inorganic nitrate/nitrite ion transport and metabolism. Although it is well documented that amino acids have the most significant effect on aphids, there is evidence that soluble nitrogen content also plays a significant role. Haltich et al., (2000) have studied the effect of nitrogen-fertilizers and apple cultivars on RAA. They concur, as with previous studies, indicating that the amount of soluble nitrogen (controlled using nitrogen fertilizers) in apple phloem varies seasonally and has an effect on aphid numbers. Of particular interest was the correlation between the drop in nitrogen in summer and aphid migration to the secondary host. The upregulation of arabinose efflux permease in aphids on apple may therefore be the result of this drop in nitrogen, the move to the secondary host plantain then seems to remove the need to express high levels of nitrate/nitrite ion transporters. This observation may add some weight to the argument that the inducer of host alternation is nitrogen availability. When the gene content of the aphid’s primary symbiont (Buchnera) was sequenced, it was found that key genes in the biosynthesis of amino acids were missing, notably isoleucine (ilvA) and methionine (MetC). Both these amino acids are unable to be produced by animals, and therefore they need to obtain these from other organisms. How the symbiont and aphid achieve this is unclear. Two differentially expressed amino acid metabolising genes have appeared in the gene expression profiles of RAA on plantain. What is intriguing is that these two genes code for homologues of the two missing enzymes; threonine dehydratase/deaminase is IlvA and cystathionine beta-lyase (cystathionase) is MetC. Further analysis is required to ascertain if this means the isoleucine and methionine biosynthesis pathways are intact in RAA. The blastx/SwissProt matches are mainly bacterial matches, suggesting the enzyme may be from the symbiont or another resident bacteria/fungus. The blastx/Swiss Prot search matches are initially sequences obtained from insect-derived libraries and then become bacterial. It is not clear if the insect matches (Drosophila, mosquito, bee) represent genuine insect sequence or comes from resident microbes. However, these two pathways might be particularly vulnerable to disruption. A recent paper (Ejim et al., 2007) has investigated the biosynthesis of methionine as an attractive antibiotic target given its importance in protein and DNA metabolism and its absence in mammals. The have performed a high-throughput screen of the cystathionine beta-lyase against a library of 50 000 small molecules and have identified several compounds that inhibit CBL enzyme activity in vitro. Antisymbiont targeted aphid control is slowly gaining credence among scientists (Le-Feuvre et al., 2007; Douglas, 2007) and are likely to yield novel targets in the future. 6.4 Candidate genes As no genes that are part of the three extensive enzyme detoxification systems (cytochrome P450s, carboxylesterases - COEs and glutathione transferases -GSTs) were found during this project, an attempt was made to see if the genes could be isolated from RAA. Using the annotated Drosophila and mosquito sequences in GenBank, aphid homologues of the enzymes were found and degenerate primer sets designed. The sequences the RAA GST and COE genes have now been successfully isolated using the same cDNA used in the differential display experiments. The P450 primers, which were the only set to prime from the polyA mRNA tail, produced too many bands to sequence, and will need further redesigning. Work can therefore begin on candidate gene work as part of our pest control studies. The GST and COE gene products will now be tested on EMR’s real-time PCR machine. 7. DISCUSSION ON FUTURE EXPLOITABLE CONTROL POSSIBILITIES This project has shown that natural resistance to RAA exist in the Malus gene pool and that these represent exploitable control possibilities. The resistance of cv. Ontario is particularly impressive and has already attracted the attention of the EMR apple breeders. It is, however, important when developing resistant cultivars to try and understand the cause of the resistance and the response to the resistance, in order to address issues of durability. This is borne out by observations in this study of the cv. Florina whose resistance is not effective in the field or gauzehouse. This study has used genome-wide gene expression profiling to monitor the transcriptional background when aphids are exposed to different hosts. Many of the genes discovered have the potential to become robust RAA stress markers to rapidly qualify and quantify the level of underlying resistance in a plant, and so dramatically reduce the timescale involved in developing aphid resistant plants. As these genes highlight stressed systems in the aphid, they also flag potentially vulnerable targets for disruption. It has been shown in this study that RAA may be particularly vulnerable to the nitrogen concentration in apple, forcing it to respond genetically by upregulating transporters and switching host. A closer look at aphid-nitrogen may yield some exploitable control possibilities. The genes characterised in the profiling of RAA on resistant hosts shows a major global metabolic change takes place involving a number of biological systems, in particular protein synthesis and energy metabolism. Of particular interest is NADH:ubiquinone dehydrogenase which is seen to be down regulated in the resistant samples. This enzyme is the first enzyme of the mitochondrial electron transport chain. It is the well known as being the target for the organic insecticide Rotenone found in several plants. These facts correlate well with the effects seen on aphids on resistant plants, and shows these pathways may be involved in apple resistance. Another gene differentially expressed is the heat shock protein dnaK from the symbiont Buchnera. This opens up another potential system of exploitable control opportunities. Antisymbiont targeted aphid control is slowly gaining interest among scientists (Le-Feuvre et al., 2007; Douglas, 2007) and are likely to yield novel targets in the future. It is notable that there were no standard detoxification genes discovered during this project. Other projects not using targeted candidate gene approaches have also not isolated detox genes as part of their hemipteran genomic profiling (Sharma et al., 2004; Francis et al., 2006) It is possible differential display may have missed these genes. It is also possible that detox genes do not play a major role in either apple resistance or host alternation (Vanhaelen and Haubruge, 2005). Now that the some RAA detox genes have been sequenced during this project it will be possible to test these genes directly. It was also shown that a switch in focus away from leaf damage (which is quantified during RAA attacks), to the real problem of fruit damage, may also lead to new exploitable control possibilities. Leaf damage may prove to be irrelevant if it is shown to be an entirely different reaction system to apple fruit damage (Dysaphis devecta, a very close congeneric species to Dysaphis plantaginea, actually produces more leaf damage but does not cause fruit damage). Here the result of the cv. MacIntosh screen on flowering plants may warrant further investigation. If indeed RAA for some reason does not feed on McIntosh flower shoots and this avoids fruit damage, then this could be another exploitable tolerance mechanism. Indeed, a change in emphasis from the current one on aphid defence by antibiosis (poisoning aphids) mechanisms to an emphasis on tolerance mechanisms (reducing the damage by the aphid) may perhaps yield a novel approach to RAA control. The next step may be to determine at the genetic and morphological level why apple fruit responds to RAA, but not to D. devecta or the other apple aphids, with a view to breeding out any genetic determinants to the RAA reaction. An added benefit of tolerance is that. unlike antibiosis, it does not exert a selection pressure on the aphid, and so resistance breakdown is unlikely to be an issue as it is in the less durable pesticide-plant toxin system of control. An unexpected discovery during this project was the identification of two viruses infecting RAA (Ryabov, 2007), which themselves offer a possible future control opportunity. The effects of these viruses on RAA require further investigation. 8. REFERENCES Arnaoudov V., Kutinkova H. (2006). Susceptibility of some apple cultivars to infestation by the rosy apple aphid (Dysaphis plantaginea pass., Homoptera: Aphididae) Journal of Fruit and Ornamental Plant Research 14: 37-142 BOUGHTON, A.J., HOOVER, K. & FELTON, G.W. (2006). Impact of chemical elicitor applications on greenhouse tomato plants and population growth of the green peach aphid, Myzus persicae. Entomologia Experimentalis et Applicata 120(3):175-188 Briggs J.B. & Alston, F.H. (1969). Sources of pest resistance in apple cultivars. Reports of East Malling Research Station 1968, pp 159-162 Cooper, W. R.; Goggin, F. L. (2005). Effects of jasmonate-induced defences in tomato on the potato aphid, Macrosiphum euphorbiae. Entomologia Experimentalis et Applicata, 115, (1), 107-115 Cross J V. & Berrie A M. (2000). Susceptibility of 42 apple varieties to pests and diseases 1998-2000. Report on Apple and Pear Research Council project SP119 issued 14 November 2000, 13 pp Cross J V. & Berrie A M. (2002). Susceptibility of 42 apple varieties to pests and diseases 2001. Report on Apple and Pear Research Council project SP119 issued 18 March 2002, 9 pp Cross J V. & Berrie A M. (2003). Susceptibility of 42 apple varieties to pests and diseases 2001. Report on Apple and Pear Research Council project SP119 issued 1 May 2003, 15 pp Cross, J.V. & Berrie, A.M. (2004). Susceptibility of apple varieties to pests and diseases, 2003. HDC Project TF119. Final Report Jerry Cross, Stella Knight, Angela Berrie, Xiangming Xu, Chris Firth & David Johnson (2005). Varieties and Integrated Pest and Disease Management for Organic Apple Production. HORTLINK Project HL0150LOF. Final Report Douglas Angela E. (2007). Symbiotic microorganisms: untapped resources for insect pest control.Trends in Biotechnology, 25(8):338-342 C.C. FIGUEROA, N. PRUNIER-LETERME, C. RISPE, F. SEPÚLVEDA, E. FUENTES-CONTRERAS, B. SABATER-MUÑOZ, J.-C. SIMON & D. TAGU (2007). Annotated expressed sequence tags and xenobiotic detoxification in the aphid Myzus persicae (Sulzer) Insect Science 14(1):29–45 Ejim L.J., Blanchard J.E., Koteva K.P., Sumerfield R., Elowe N.H., Chechetto J.D., Brown E.D., Junop M.S. & Wright G.D. (2007). Inhibitors of bacterial cystathionine beta-lyase: leads for new antimicrobial agents and probes of enzyme structure and function. J Med. Chem.; 50(4):755-64 FEDER, M.E. & WALSER, J.-C. (2005). The biological limitations of transcriptomics in elucidating stress and stress responses. Journal of Evolutionary Biology, 18(4):901-910 FORREST J.M.S. & A.F.G. DIXON (1975). The induction of leaf-roll galls by the apple aphids Dysaphis devecta and D. Plantaginea. Annals of Applied Biology 81(3)281–288 Francis F, Gerkens P, Harmel N, Mazzucchelli G, De Pauw E, Haubruge E. (2006) Proteomics in Myzus persicae: effect of aphid host plant switch. Insect Biochem Mol Biol.,36(3):219-27 Haltrich A., Papp J., Fail J., Kis L. (2000). Effect of nitrogen-fertilizers and apple cultivars on aphids under IPM treatment conditions. Proc. of the Int. Conf. on Integrated Fruit Production. Acta Hort. 525:209-216 Harmel N., E. Létocart, A. Cherqui, P. Giordanengo, G. Mazzucchelli, F. Guillonneau, E. De Pauw, E. Haubruge & F. Francis (2008). Identification of aphid salivary proteins: a proteomic investigation of Myzus persicae. Insect Molecular Biology 17(2):165–174 Harvey N.G., J.D. Fitzgerald, C.M. James, M.G. Solomon. (2003). Isolation of microsatellite markers from the rosy apple aphid, Dysaphis plantaginea. Molecular Ecology Notes, 3:111-112 Hunter, W.B., Dang, P.M., Bausher, M.G., Chaparro, J.X., McKendree, W., Shatters R.G. Jr, McKenzie, C.L. & Sinisterra, X.H. (2003). Aphid biology: expressed genes from alate Toxoptera citricida, the brown citrus aphid. Journal of Insect Science 3:23 Kehr Julia (2006). Phloem sap proteins: their identities and potential roles in the interaction between plants and phloem-feeding insects; Journal of Experimental Botany 57(4):767-74 Le-Feuvre, R.R. Ramírez, C.C.; Olea, N. & Meza-Basso, L. (2007) Effect of the antimicrobial peptide indolicidin on the green peach aphid Myzus persicae (Sulzer). Journal of Applied Entomology, 131(2):71-75 Lyth M., 1985. Hypersensitivity in apple to feeding by Dysaphis-plantaginea: effects on aphid biology Annals of Applied Biology 107(2):155-161 Miñarro, M. & Dapena, E. (2004). Sustainable control of the rosy apple aphid Dysaphis plantaginea (póster) 6th Internacional Conference on Integrated Fruit Production. Baselga di Piné (Italia). Septembre 2004 Morgan & Richards (2002) The New Book of Apples, Ebury Press 2002 Ngeli G. & Simoni S. (2006). Apple cultivars acceptance by Dysaphis plantaginea Passerini (Homoptera: Aphididae) Journal of Pest Science 79(3)175-179 Qubbaj, T. Reineke & A. Zebitz, C.P.W. (2005). Molecular interactions between rosy apple aphids, Dysaphis plantaginea, and resistant and susceptible cultivars of its primary host Malus domestica Entomologia Experimentalis et Applicata, 115:145-152 Radoslav Andreev & Hristina Kutinkova (2004). Resistance to aphids and scale insects in nine apple cultivars. Journal of Fruit and Ornamental Plant Research 12 Special ed. 216-221 Ramsey J.S., Wilson A.C., de Vos M., Sun Q., Tamborindeguy C., Winfield A., Malloch G., Smith D.M., Fenton B., Gray S.M. & Jander G. (2007). Genomic resources for Myzus persicae: EST sequencing, SNP identification, and microarray design. BMC Genomics. 8:423 Rat-Morris E., Crowther S. & Guessoum M. (1999). Resistance-breaking biotypes of rosy apple aphid Dysaphis plantaginea on resistant cv. Florina. IOBC/WPRS Bulletin 22(10):71-76 Ryabov, J. (2007). A novel virus isolated from the aphid Brevicoryne brassicae with similarity to Hymenoptera. Genetics of Virology 88:2590-2595 Sabater-Muñoz, B., Legeai, F., Rispe, C., Bonhomme, J., Dearden, P., Dossat, C., Duclert, A., Gauthier, J.P., Ducray, D.G., Hunter, W., Dang, P., Kambhampati, S., Martinez-Torres, D., Cortes, T., Moya, A., Nakabachi, A., Philippe, C., Prunier-Leterme, N., Rahbé, Y., Simon, J.C., Stern, D.L., Wincker, P. & Tagu, D. (2006) Large-scale gene discovery in the pea aphid Acyrthosiphon pisum (Hemiptera). Genome Biology 7(3):R21 Sharma, R., Komatsu, S., Noda, H. (2004). Proteomic analysis of brown planthopper: application to the study of carbamate toxicity. Insect Biochemicals and Molecular Biology 34(5):425-32 Shigenobu, S., Watanabe, H., Hattori, M., Sakaki, Y. & Ishikawa, H. (2000). Genome sequence of the endocellular bacterial symbiont of aphids Buchnera sp. APS. Nature 407(6800):81-6 Smith, C. Michael, Boyko, Elena V. ( 2007). The molecular bases of plant resistance and defence responses to aphid feeding: current status. Entomologia Experimentalis et Applicata, 122(1), 1-16 Tobutt, Kenneth R. (Maidstone, GB2) Columnar apple tree --Telamon variety. United States Patent PP06224 Vanhaelen Francis, F., N., Haubruge, E. (2005). Glutathione S-transferases in the adaptation to plant secondary metabolites in the Myzus persicae aphid. Archives of Insect Biochemistry and Physiology 58(3):166-174 Voelckel, C., Weisser, W. W., Baldwin, I. T. (2004). An analysis of plant-aphid interactions by different microarray hybridization strategies. Molecular Ecology, 13:3187-3195 Wilson, Alex C.C., Helen, E. Dunbar, Gregory K. Davis, Wayne B. Hunter, David L. Stern & Nancy A. Moran (2006). A dual-genome microarray for the pea aphid, Acyrthosiphon pisum, and its obligate bacterial symbiont, Buchnera aphidicola. BMC Genomics 7: 50 Zhu-Salzman, Keyan, Ron A. Salzman, Ji-Eun Ahn, & Hisashi Koiwa (2004). Transcriptional Regulation of Sorghum Defence Determinants against a Phloem-Feeding Aphid. Plant Physiology 134: 420-431 Zhu-Salzman, K., D.S. Luthe, & G.W. Felton (2008). Arthropod-Inducible Proteins: Broad Spectrum Defences against Multiple Herbivores. Plant Physiology 146(3):852 - 858 9 ACTIONS ARISING FROM RESEARCH Crosses have been made by EMR’s apple breeders with three of the cultivars of interest in this project, in order to begin mapping resistance inheritance. All the crosses were with the susceptible columnar cv. Telemon (Tobutt). The crosses were Malus robusta 3760, Florina and Ontario. These progeny have been scored after artificially inoculating them with RAA in 2006 and 2007. Since this project has determined an unexpected lack of resistance in Florina in the UK, it was not surprising to find all the progeny were susceptible and therefore uninformative. The Ontario cross was scored as 32 susceptible, 15 intermediate and 7 resistant; a 4:2:1 ratio. The 3760 cross was more complicated. Resistance in 3760 was traditionally scored as a hypersensitive shoot tip death resulting in no aphid growth. This extensive damage is why this resistance has not been exploited in breeding. We have scored them as resistant (tip dead, no aphids) 13, benign resistant (tip alive, no aphids) 17, and susceptible (many aphids, tip alive, and usually leaf curl) 32. This may indicate that a resistance mechanism could be usable without confounding hypersensitive tip death. Now that potential resistance markers have been identified to monitor an RAA’s stress on a plant, these apple crosses will prove useful in testing which biomarkers are best; by matching biomarker expression with visual colony scoring. The best markers can then be used to rapidly qualify and quantify the level of underlying resistance in a plant, and so dramatically reduce the timescale involved in developing aphid resistant plants.
9. This section should be used to record links (hypertext links where possible) or references to other published material generated by, or relating to this project. | |||||||||||||||||||||||||||||||||||||||||||
