Identification and cloning of three endogenous genes that may confer resistance to pathogens in citrus including CLas and CTV

Pedro Iván Ventura-Medina, María Alejandra Gutiérrez-Espinosa, Vicente Febres, Gustavo Mora-Aguilera, Alejandrina Robledo-Paz

Abstract


Candidatus Liberibacter asiaticus (CLas), causal agent of Huanglongbing disease (HLB), and the Citrus tristeza virus (CTV) constitute a serious threat to Mexican citriculture because its endemic status in limes in several regions of the Pacific coast and also for the recent detection of severe strains, respectively. The identification and cloning of some genes related to the Systemic Acquired Resistance (SAR) of the plant could help to confront the diseases as an integrated management strategy. This work had the objective of cloning specific genes inherent to the plant as a first step to obtain transgenic citrus plants putatively resistant to CLas. The genes were selected based on the response of citrus plants to a wide range of pathogens, they were; a regulating gene for salicylic acid signaling (SA) as the inducer gen Azelaic Acid Induced 1 (AZI-1), a Citrus NonRace-Specific Disease Resistance 1 (CsNDR-1) and a pathogenesis related protein 1 gen (PR-1). These three genes (AZI-1, PR-1 and CsNDR-1) were independently inserted into pUC118-FMVPoly-2-1, subsequently they were subcloned individually into the vector pCAMBIA 2301 while gene AZI-1 was only cloned into pCAMBIA 2201. The CsNDR-1 construct was cloned into Agrobacterium tumefaciens strains EHA5 and AgL1 while genes AZI-1 and PR-1 could not be inserted into these strains of Agrobacterium. These constructions are the first step to be able to generate transgenic citrus as an alternative to face the HLB.


Keywords


SAR; plasmids; AZI-1; PR-1; CsNDR-1

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References


Almeida AB, Mourão-Filho AA, Januzzi-Mendes BM, Pavan A. y Martinelli-Rodriguez AP. 2003. Agrobacterium-mediated transformation of Citrus sinensis and Citrus limonia epicotyl segments. Scientia Agricola 60:1: 23-29. http:// dx.doi.org/10.1590/S0103-90162003000100005.

Belkhadir Y, Nimchuk Z, Hubert D, Mackey D and Danglet J. 2004. Arabidopsis RIN4 negatively regulates disease resistance mediated by RPS2 and RPM1 downstream or independent of the NDR1 signal modulator and is not required for the virulence functions of bacterial type III effectors AvrRpt2 or AvrRpm1. The Plant Cell. 16:10: 2822–2835. https://doi.org/10.1105/tpc.104.024117

Bové JM. 2012. Huanglongbing and the future of citrus in Sao Paulo, Brazil. Journal of Plant Pathology. 94(3):465-467.

Da Graca JV, Douhan GW, Halbert SE, Keremane ML, Lee RF, Vidalakis G, and Zhao H. 2016. Huanglongbing: An overview of a complex pathosystem ravaging the world’s citrus. Journal of Integrative Plant Biology 58(4):373-387. https://doi.org/10.1111/jipb.12437

De Oliveira MLP, Stover ED and Thomson JG 2015. The codA gene as a negative selection marker in Citrus. SpringerPlus 4:264: 3-7. https://doi.org/10.1186/s40064-015-1047-y

Ding F, Jin S, Hong N, Zhong Y and Cao Q. 2008. Vitrification–cryopreservation, an efficient method for eliminating Candidatus Liberobacter asiaticus, the citrus Huanglongbing pathogen, from in vitro adult shoot tips. Plant Cell Reports 27(2): 241–250. https://doi.org/10.1007/s00299007-0467-8

Doyle JJ and Doyle JL. 1987. A rapid DNA isolation procedure for small quantities of small quantities of fresh leaf tissue. Phytochemical bulletin 19. 11-15

Dutt M, Barthe G, Irey M and Grosser J. 2015. Transgenic citrus expressing an Arabidopsis NPR-1 gene exhibit enhanced resistance against Huanglongbing (HLB; Citrus Greening). Public Library of Science One. 10:9: 1-17. https:// doi.org/10.1371/journal.pone.0137134

Febres VJ, Niblet CL, Lee RF and Moore GA. 2003. Characterization of grapefruit plants (Citrus paradisi Macf.) transformed with citrus tristeza closterovirus genes. Plant Cell Reports 21: 421-428. https://doi.org/10.1007/s00299002-0528-y

Fu S, Shao J, Zhou Ch, and Hartung JS. 2016. Transcriptome analysis of sweet orange trees infected with ‘Candidatus Liberibacter asiaticus’ and two strains of Citrus Tristeza Virus. BMC Genomics 17:349 https://doi.org/10.1186/ s12864-016-2663-9.

Gao QM, Zhu S, Kachroo P and Kachoroo A. 2015. Signal regulators of systemic acquired resistance. Frontiers in Plant Science 6: 228. https://doi.org/10.3389/fpls.2015.00228

Gómez S y Mejía Z. 2011. Respuesta de hipersensibilidad, una muerte celular programada para defenderse del ataque por fitopatógenos. Revista Mexicana de Fitopatología. 29:2: 154-164. Disponible en línea: http://www.redalyc.org/ pdf/612/61222864007.pdf

Gottwald TR. 2007. Citrus canker and citrus huanglongbing, two exotic bacterial diseases threatening the citrus industries of the western hemisphere. Outlooks on PestManagement 18(6):274–279. https://doi.org/10.1564/18dec09

Hajeri S, Killiny N, El-Mohtar C, Dawson WO and Gowda S. 2014. Citrus tristeza virus-based RNAi in citrus plants induces gene silencing in Diaphorina citri, a phloem-sap sucking insect vector of citrus greening disease (Huanglongbing). Journal of Biotechnology 176:20: 42–49. http://doi.org/10.1016/j.jbiotec.2014.02.010

Hall DG, Richardson ML, Ammar El-Desouky and Halbert SE. 2012. Asian citrus psyllid, Diaphorina citri, vector of citrus huanglongbing disease. Entomologia Experimentalis et Applicata. 146: 207–223. 10.1111/eea.12025

Hao G, Stover E and Gupta G. 2016. Overexpression of a modified plant thionin enhances disease resistance to Citrus canker and Huanglongbing (HLB). Frontiers in Plant Science 7: 1078: 1-11. https://doi.org/10.3389/ fpls.2016.01078

Hoffman MT, Doud MS, Williams L, Zhang M and Ding F. 2012. Heat treatment eliminates ‘Candidatus Liberibacter asiaticus’ from infected citrus trees under controlled conditions. Phytopathology. 103:15–22. https://doi.org/10.1094/ PHYTO-06-12-0138-r

Hu Y, Zhong X, Liu X, Lou B, Zhou Ch, and Wang X. 2017. Comparative transcriptome analysis unveils the tolerance mechanisms of Citrus hystrix in response to ‘Candidatus Liberibacter asiaticus’ infection. Public Library of Science ONE 12(12)e0189229. https://doi.org/a0.1371/journal. pone.0189229.

Ichinose K, Miyazi K, Matsuhira K, Yasuda K, Sadoyama Y, Do HT and Doan VB. 2010. Unreliable pesticide control of the vector psyllid Diaphorina citri (Hemiptera: Psyllidae) for the reduction of microorganism disease transmission. Journal of Environmental Science and Health B. 45:5:466– 472. https://doi.org/10.1080/03601231003800263

Kayim M and Koc NK. 2005. Improved transformation efficiency in Citrus by plasmolysis treatment. Journal of Plant Biochemistry and Biotechnology 14(1):15-20. https://doi. org/10.1007/BF03263218

Lu H, Zhang C, Albrecht U, Shimizu R, Wang G and Bowman KD. 2013. Overexpression of a citrus NDR1 ortholog increases disease resistance in Arabidopsis. Frontiers in Plant Science. 4:157:1-10. https://doi.org/10.3389/ fpls.2013.00157

Mafra V, Martins P, Francisco C, Ribeiro-Alves M, FreitasAstúa J and Machado M. 2013. Candidatus Liberibacter americanus induces significant reprogramming of the transcriptome of the susceptible citrus genotype. BMC Genomics. 14:247. https://doi.org/10.1186/1471-2164-14247.

Mora-Aguilera G, Robles GP, López AJL, Flores SJ, Acevedo SG, Domínguez MS, Gutiérrez EA and Loeza KE. 2014. Current situation and perspectives in management of citrus HLB. Mexican Journal of Phytopathology 32(2):108-119. https://doi.org/10.7550/rmb.46314

Nishimura MT and Dangl JL. 2010. Arabidopsis and the plant immune system. The Plant Journal 61(6): 1053–1066. https://doi.org/10.1111/j.1365-313X.2010.04131.x

Pajerowska MK, Emerine DK and Shahid-Mukhtar M. 2013. Tell me more: roles of NPRs in plant immunity. Trends in Plant Science. 18:7: 402-4011. https://doi.org/10.1016/j. tplants.2013.04.004

Pinheiro TT, Figueira A and Latado RR. 2014. Early-flowering sweet orange mutant “x11” as a model for functional genomic studies of Citrus. BMC Research Notes. 7:511: 1-7. https://doi.org/10.1186/1756-0500-7-511.

Rawat N, Kumar B, Albrecht U, Du D, Huang M, Yu Q, Zhang Y, Duan YP, Bowman KD, Gmitter FGJr, and Deng Z. 2017. Genome resequencing and transcriptome profiling reveal structural diversity and expression patterns of constitutive disease resistance genes in Huanglongbing-tolerant Poncirus trifoliate and its hybrids. Horticulture Research 4,17064. https://doi.org/10.1038/hortres.2017.64

Shi Q, Febres VJ, Jones JB and Moore GA. 2016. A survey of FLS2 genes from multiple citrus species identifies candidates for enhancing disease resistance to Xanthomonas citri ssp. citri. Horticultural Research 3:16022: 1-11. https:// doi.org/10.1038/hortres.2016.22.

Van-Loon LC and Van Strien EA. 1999. The families of pathogenesis-related proteins, their activities, and comparative analysis of PR-1 type proteins. Physiological and Molecular Plant Pathology. 55. 85–97. https://doi.org/10.1006/ pmpp.1999.0213

Vazquez-García M, Velázquez-Monreal J, Medina-Urrutia VM, Cruz-Vargas CD, Sandoval-Salazar M, Virgen-Calleros G and Torres-Moran JP. 2013. Insecticide resistance in adult Diaphorina citri Kuwayama from lime orchards in Central West Mexico. Southwestern Entomologist. 38: 579-596. http://doi.org/10.3958/059.038.0404

Yu K, Moreira-Soares J, Kumar-Mandal M, Wang C, Chanda B, Gifford AN, Fowler JS, Navarre D, Kachroo A and Kachroo P. 2013. A feedback regulatory loop between G3P and lipid transfer proteins DIR1 and AZI-1 mediates AzalaicAcid-induced system immunity. Cell Reports. 3(4):12661278. http://doi.org/10.1016/j.celrep.2013.03.030

Zhang M, Guo Y, Powell C, Doud M, Yang C and Duan Y. 2014. Effective antibiotics against ‘Candidatus Liberibacter asiaticus’ in HLB-affected Citrus plants identified via the graft-based evaluation. Public Library of Science One. 9:11: e111032. https://doi.org/10.1371/journal. pone.0111032

Zhang M, Powell CA, Guo Y, Doud MS and Duan Y. 2012 A graft-based chemotherapy method for screening effective molecules and rescuing huanglongbing-affected citrus plants. Phytopathology. 102(6): 567–574. https://doi. org/10.1094/PHYTO-09-11-0265




DOI: http://dx.doi.org/10.18781/R.MEX.FIT.1906-6

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