Abstract
RNA interference (RNAi) has proved a powerful genetic tool for silencing genes in plants. Host-induced gene silencing of pathogen genes has provided a gene knockout strategy for a wide range of biotechnological applications. The RXLR effector Avr3a gene is largely responsible for virulence of oomycete plant pathogen Phytophthora infestans. In this study, we attempted to silence the Avr3a gene of P. infestans through RNAi technology. The P. infestans inoculation resulted in lower disease progression and a reduction in pathogen load, as demonstrated by disease scoring and quantification of pathogen biomass in terms of Pi08 repetitive elements, respectively. Transgenic plants induced moderate silencing of Avr3a, and the presence and/or expression of small interfering RNAs, as determined through Northern hybridization, indicated siRNA targeted against Avr3a conferred moderate resistance to P. infestans. The single effector gene did not provide complete resistance against P. infestans. Although the Avr3a effector gene could confer moderate resistance, for complete resistance, the cumulative effect of effector genes in addition to Avr3a needs to be considered. In this study, we demonstrated that host-induced RNAi is an effective strategy for functional genomics in oomycetes.
Similar content being viewed by others
References
Ah Fong AMV, Bormann-Chung CA, Judelson HS (2008) Optimization of transgene-mediated silencing in Phytophthora infestans and its association with small interfering RNAs. Fungal Genet Biol 45:1197–1205
Baum JA, Bogaert T, Clinton W, Heck GR, Feldmann P, Ilagan O et al (2007) Control of coleopteran insect pests through RNA interference. Nat Biotechnol 25:1322–1326
Birch PR, Armstrong M, Bos J, Boevink P, Gilroy EM, Taylor RM, Wawra S, Pritchard L, Conti L, Ewan R, Whisson S, van West P, Sadanandom A, Kamoun S (2009) Towards understanding the virulence functions of RXLR effectors of oomycetes plant pathogen Phytophthora infestans. J Exp Bot 60:1133–1140
Bos JIB, Kanneganti TD, Young C, Cakir C, Huitema E, Win J, Armstrong MR, Birch PR, Kamoun S (2006) The C-terminal half of Phytophthora infestans RXLR effector Avr3a is sufficient to trigger R3a-mediated hypersensitivity and suppress INF1-induced cell death in Nicotiana benthamiana. Plant J 48:165–176
Bos JI, Armstrong MR, Gilroy EM, Boevink PC, Hein I, Taylor RM, Zhendong T, Engelhardt S, Vetukuri RR, Harrower B, Dixelius C, Bryan G, Sadanandom A, Whisson SC, Kamoun S, Birch PR (2010) Phytophthora infestans effector Avr3a is essential for virulence and manipulates plant immunity by stabilizing host E3 ligase CMPG1. Proc Natl Acad Sci U S A 107:9909–9914
Bos JI, Chaparro-Garcia A, Quesada-Ocampo LM, McSpadden Gardener BB, Kamoun S (2009) Distinct amino acids of the Phytophthora infestans effector Avr3a condition activation of R3a hypersensitivity and suppression of cell death. Mol Plant Microbe Interact 22:269–281
Catanzariti AM, Dodds PN, Lawrence GJ, Agliffe MA, Ellis JG (2006) Haustorially expressed secreted proteins from flax rust are highly enriched for avirulence elicitors. Plant Cell 18:243–256
Chapman S, Laura JS, Petra CB, Stefan E, Colin JA, Brian H, Nicolas C, McGeachy K, Pauline SMV, Xinwei C, Paul RJ B, Ingo H (2014) Detection of the virulent form of Avr3a from Phytophthora infestans following artificial evolution of potato resistance gene R3a. Plos One 9:1–11
Caten CE, Jinks JL (1968) Spontaneous variability of single isolates of Phytophthora infestans I. Cultural variation. Can J Bot 46:329–347
Chen H, Nelson RS, Sherwood JL (1994) Enhanced recovery of transformants of Agrobacterium tumefaciens after freeze-thaw transformation and drug selection. BioTechn 16:664–670
Gomes NP, Bjerke G, Llorente B, Szostek SA, Emerson BM, Espinosa JM (2006) Gene specific requirement for P-TEFB activity and RNA polymerase II phosphorylation within the p53 transcriptional program. Genes Dev 20:601–612
Hass B et al (2009) The genome sequence of the Irish famine pathogen Phytophthora infestans. Nature 461:393–398
Haverkort AJ, Boonekamp PM, Hutten R, Jacobsen E, Lotz LAP, Kessel GJT et al (2008) Societal costs of late blight in potato and prospects of durable resistance through cisgenic modification. Potato Res 51:47–57
Haverkort AJ, Struik P, Visser R, Jacobsen E (2009) Applied biotechnology to combat late blight in potato caused by Phytophthora infestans. Potato Res 52:249–264
Hood EE, Gelvin SB, Melchers LS, Hoekema A (1993) New Agrobacterium helper plasmids for gene transfer to plants. Trans Res 2:208–218
Judelson HS, Tooley PW (2000) Enhanced polymerase chain reaction methods for detecting and quantifying Phytophthora infestans in plants. Phytopathology 90:1112–1119
Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔCt method. Methods 25:402–408
Llorente B, Bravo-Almonacid F, Cvitanich C, Orlowska E, Torres HN, Flawiá MM, Alonso GD (2010) A quantitative real-time PCR method for in planta monitoring of Phytophthora infestans growth. Lett Appl Microbiol 51:603–610
Malcolmson JF (1976) Assessment of field resistance to blight (Phytophthora infestans) in potatoes. Trans Br Mycol Soc 67:321–325
Mansoor S, Zafar Y, Briddon RW (2006) Geminivirus disease complexes: the threat is spreading. Trends Plant Sci 11:209–212
Mao YB, Cai W, Wang J, Tao X, Wang L et al (2007) Silencing a cotton bollworm P450 monooxygenase gene by plant-mediated RNAi impairs larval tolerance of gossypol. Nat Biotechnol 25:1307–1313
Mathieu R, Bender J (2004) RNA-directed DNA methylation. J Cell Sci 117:4881–4888
Niblett CL, Bailey AM (2012) Potential applications of gene silencing or RNA interference (RNAi) to control disease and insect pests of date palm. Emir J Food Agric 24:462–469
Nowara D, Gay A, Lacomme C, Shaw J, Ridout C et al (2010) HIGS: host-induced gene silencing in the obligate biotrophic fungal pathogen Blumeria graminis. Plant Cell 22:3130–3141
Nunes ACS, Vianna GR, Cuneo F, Amaya-Farfan J, de Capdeville G, Rech EL, Aragao FJL (2006) RNAi mediated silencing of the myo-inositol-1-phosphate synthase gene (GmMIPS1) in transgenic soybean inhibited seed development and reduced phytate content. Planta 224:125–132
Panwar V, McCallum B, Bakkeren G (2013a) Endogenous silencing of Puccinia triticina pathogenicity genes through in planta expressed sequences leads to suppression of rust diseases on wheat. Plant J 73:521–532
Panwar V, McCallum B, Bakkeren G (2013b) Host-induced gene silencing of wheat leaf rust fungus Puccinia triticina pathogenicity genes mediated by the Barley stripe mosaic virus. Plant Mol Biol 81:595–608
Pattanayak D, Agarwal S, Sumathi S, Chakrabarti SK, Naik PS, Khurana SMP (2005) Small but mighty RNA-mediated inference in plants. Indian J Exp Biol 43:7–24
Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual, 2nd edn. Cold Spring Harbour Press, USA
Smith NA, Singh SP, Wang MB, Stoutjesdijk PA, Green AG, Waterhouse PM (2000) Gene expression: total silencing by intron-spliced hairpin. Nature 407:319–320
Vance V, Vaucheret H (2001) RNA silencing in plants—defense and counterdefense. Science 292:2277–2280
Voinnet O (2005) Non-cell autonomous RNA silencing. FEBS Lett 579:5858–5871
Wang MB, Waterhouse PM (2001) Application of gene silencing in plants. Curr Opin Plant Biol 5:146–150
Wesley SV et al (2001) Construct design for efficient, effective and high throughput gene silencing in plants. Plant J 27:581–590
Whisson SC et al (2007) A translocation signal for delivery of oomycetes effector proteins inside host plant cells. Nature 450:115–118
Whisson SC, Vetukuri RR, Avrova AO, Dixelius C (2012) Can silencing of transposons contribute to variation in effector gene expression in Phytophthora infestans? Mob Genet Elem 2:110–114
Yin C, Jurgenson JE, Hulbert SH (2011) Development of a host-induced RNAi system in the wheat stripe rust fungus Puccinia striiformis f. sp. tritici. Mol Plant Microbe Interact 24:554–561
Yongli Q, Lin L, Qin X, Cristina F, James W, Jinxia S, Xianbing W, Xigang L, Qijun X, Shushu J, Fuchun Z, Yuanchao W, Howard SJ, Xuemei C, Wenbo M (2013) Oomycete pathogens encode RNA silencing suppressors. Nature 45:330–335
Acknowledgments
We are thankful to the Phytophthora, Fusarium, and Ralstonia Disease of Horticultural Crops Project (PhytoFuRa), Indian Council of Agricultural Research, New Delhi, for their financial support.
Conflict of interest
The authors declare that they have no conflict of interest.
Author information
Authors and Affiliations
Corresponding author
Electronic supplementary material
Below is the link to the electronic supplementary material.
ESM 1
(DOCX 154 kb)
Rights and permissions
About this article
Cite this article
Sanju, S., Siddappa, S., Thakur, A. et al. Host-mediated gene silencing of a single effector gene from the potato pathogen Phytophthora infestans imparts partial resistance to late blight disease. Funct Integr Genomics 15, 697–706 (2015). https://doi.org/10.1007/s10142-015-0446-z
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s10142-015-0446-z