Location: Subtropical Plant Pathology ResearchTitle: Molecular mechanisms behind the accumulation of adenosine triphosphate (ATP) and H2O2 in citrus plants in response to ‘Candidatus Liberibacter asiaticus’ infection
Submitted to: Horticulture Research
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 7/5/2017
Publication Date: 8/23/2017
Citation: Pitino, M., Armstrong, C.M., Duan, Y. 2017. Molecular mechanisms behind the accumulation of adenosine triphosphate (ATP) and H2O2 in citrus plants in response to ‘Candidatus Liberibacter asiaticus’ infection. Horticulture Research. 4(Article number 17040). https://doi.org/10.1038/hortres.2017.40.
Interpretive Summary: Citrus Huanglongbing (HLB, also known as citrus greening) is the most devastating disease of citrus in the world. HLB has already caused substantial economic losses to citrus producers around the globe and threatens the citrus industry in both Florida, USA and Sao Paulo, Brazil; the top two producers of both oranges and juice in the world. HLB is associated with three different phloem-restricted, uncultured species of a-proteobacteria in the genus Candidatus Liberibacter. The species known as Candidatus Liberibacter asiaticus (Las) is the most widely distributed species in the world and the only one present in the state of Florida. Our lab was the first to release the completed genome sequence of Las and the first to attempt to characterize and locate putative Las effectors. We have since become one of the leaders in HLB research by contributing information concerning host-pathogen interactions and bacterial diversity to the field for almost a decade now. Bacteria deploy effector proteins with varying activities in order to manipulate host physiology and control pathways that regulate the ability of the host cell to propagate infection. Recently, it was discovered that Las bacteria encode effector proteins, which can be delivered into host cells. With this project we demonstrated that Las manipulates important aspects involved with HLB by increasing the host's energy supply and detoxifying the H2O2 that is accumulated in response to biotic stress, a critical step for bacterial survival in plants. Our data suggest that Las increases the levels of adenosine triphosphate (ATP) in host plants in order to increase its availability and allow for uptake through the ATP translocase previously revealed. Gene expression analysis showed an up-regulation of ATP synthase together with increase in the ATP concentration in Las infected tissues compared to non-Las infected tissues. We also demonstrated that the H2O2 production, triggered by Las infection along with the upregulation of Rboh, was manipulated by Las by altering the enzymatic mechanisms that reduce the toxic effect of H2O2. Our data show that H2O2 detoxification genes were down regulated in Las-infected tissue, suggesting that the toxic effects of H2O2 become potentially more destructive for the plant tissue and possibly induce the dramatic HLB symptoms in citrus rather than directly inhibiting Las bacteria growth. The active peroxidase encoded for by Las provides an additional line of defense to protect the bacteria against the H2O2 generated by the infected plant cell. Together, these results provide new insight into understanding the mechanisms of plant-microbial interactions during Las infection and pathogenicity mechanisms.
Technical Abstract: Candidatus Liberibacter asiaticus (Las) is a fastidious, phloem-restricted pathogen with a significantly reduced genome, and attacks all citrus species with no immune cultivars documented to date. Like other plant bacterial pathogens, Las deploys effector proteins into the organelles of plant cells, such as mitochondria and chloroplasts to manipulate host immunity and physiology. These organelles are responsible for the synthesis of adenosine triphosphate (ATP) and have a critical role in plant immune signaling during hydrogen peroxide (H2O2) production. In this study, we investigated H2O2 and ATP accumulation in relation to HLB disease in addition to revealing the expression profiles of genes critical for the production and detoxification of H2O2 and ATP synthesis. We also found that as ATP and H2O2 concentrations increased in the infected leaves so did the severity of the HLB symptoms, a trend that remained consistent amongst the four different citrus varieties tested. We have revealed a correlation between Las infection and accumulation of H2O2 and ATP in citrus leaves. Our results showed that accumulation of H2O2 and ATP increased as HLB symptoms became more pronounced on citrus leaves. The upregulation of ATP synthase, a key enzyme for energy conversion, may contribute to the accumulation of ATP in infected tissues while down-regulation of the H2O2 detoxification system may cause oxidative damage to plant macromolecules and cell structures. This may explain the origin of some of the HLB symptoms such as chlorosis or leaf discoloration. The findings in this study highlight important molecular and physiological mechanisms involved in the host plants’ response to Las infection and provide new targets for interrupting the disease cycle.