Abstract
Main conclusion
Host-derived suppression of nematode essential genes decreases reproduction of Meloidogyne incognita in cotton.
Abstract
Root-knot nematodes (RKN) represent one of the most damaging plant-parasitic nematode genera worldwide. RNAi-mediated suppression of essential nematode genes provides a novel biotechnological strategy for the development of sustainable pest-control methods. Here, we used a Host Induced Gene Silencing (HIGS) approach by stacking dsRNA sequences into a T-DNA construct to target three essential RKN genes: cysteine protease (Mi-cpl), isocitrate lyase (Mi-icl), and splicing factor (Mi-sf), called dsMinc1, driven by the pUceS8.3 constitutive soybean promoter. Transgenic dsMinc1-T4 plants infected with Meloidogyne incognita showed a significant reduction in gall formation (57–64%) and egg masses production (58–67%), as well as in the estimated reproduction factor (60–78%), compared with the susceptible non-transgenic cultivar. Galls of the RNAi lines are smaller than the wild-type (WT) plants, whose root systems exhibited multiple well-developed root swellings. Transcript levels of the three RKN-targeted genes decreased 13- to 40-fold in nematodes from transgenic cotton galls, compared with those from control WT galls. Finally, the development of non-feeding males in transgenic plants was 2–6 times higher than in WT plants, indicating a stressful environment for nematode development after RKN gene silencing. Data strongly support that HIGS of essential RKN genes is an effective strategy to improve cotton plant tolerance. This study presents the first application of dsRNA sequences to target multiple genes to promote M. incognita tolerance in cotton without phenotypic penalty in transgenic plants.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
Availability of data and materials
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
Abbreviations
- DAI:
-
Days after inoculation
- GC:
-
Giant cell
- HIGS:
-
Host Induced Gene Silencing
- Mi-cpl :
-
Cysteine protease
- Mi-icl :
-
Isocitrate lyase
- Mi-sf :
-
Splicing factor
- NG:
-
Number of galls
- PDK:
-
Pyruvate dehydrogenase kinase
- ppJ2:
-
Second-stage pre-parasitic juveniles
- RKN:
-
Root-knot nematodes
References
Abad P, Gouzy J, Aury JM et al (2008) Genome sequence of the metazoan plant-parasitic nematode Meloidogyne incognita. Nat Biotechnol 26:909–915. https://doi.org/10.1038/nbt.1482
Anjam MS, Shah SJ, Matera C et al (2020) Host factors influence the sex of nematodes parasitizing roots of Arabidopsis thaliana. Plant Cell Environ 43:1160–1174. https://doi.org/10.1111/pce.13728
Aragão FJL, Sarokin L, Vianna GR, Rech EL (2000) Selection of transgenic meristematic cells utilizing a herbicidal molecule results in the recovery of fertile transgenic soybean [Glycine max (L.) Merril] plants at a high frequency. Theor Appl Genet 101:1–6. https://doi.org/10.1007/s001220051441
Arnhold E (2013) Package in the R environment for analysis of variance and complementary analyses. Braz J Vet Res Anim Sci 50:488–492
Bakhetia M, Urwin PE, Atkinson HJ (2008) Characterisation by RNAi of pioneer genes expressed in the dorsal pharyngeal gland cell of Heterodera glycines and the effects of combinatorial RNAi. Int J Parasitol 38:1589–1597. https://doi.org/10.1016/j.ijpara.2008.05.003
Banakar P, Hada A, Papolu PK, Rao U (2020) Simultaneous RNAi knockdown of three FMRFamide-Like peptide genes, Mi-flp1, Mi-flp12, and Mi-flp18 provides resistance to root-knot nematode, Meloidogyne incognita. Front Microbiol 11:1–17. https://doi.org/10.3389/fmicb.2020.573916
Banora MY, Rodiuc N, Baldacci-Cresp F et al (2011) Feeding cells induced by phytoparasitic nematodes require γ-tubulin ring complex for microtubule reorganization. PLoS Pathog 7:e1002343. https://doi.org/10.1371/journal.ppat.1002343
Bird DMK, Kaloshian I (2003) Are roots special? Nematodes have their say. Physiol Mol Plant Pathol 62:115–123. https://doi.org/10.1016/S0885-5765(03)00045-6
Bird DM, Opperman CH (1998) Caenorhabditis elegans: a genetic guide to parasitic nematode biology. J Nematol 30:299–308
Black DL (2000) Protein diversity from alternative minireview splicing: a challenge for bioinformatics and post-genome biology. Cell 103:367–370. https://doi.org/10.1016/s0092-8674(00)00128-8
Ceccon CC, Caverzan A, Margis R et al (2020) Gene stacking as a strategy to confer characteristics of agronomic importance in plants by genetic engineering. Cienc Rural 50:1–12. https://doi.org/10.1590/0103-8478cr20190207
Charlton WL, Harel HYM, Bakhetia M et al (2010) Additive effects of plant expressed double-stranded RNAs on root-knot nematode development. Int J Parasitol 40:855–864. https://doi.org/10.1016/j.ijpara.2010.01.003
Chaudhary S, Dutta TK, Tyagi N et al (2019) Host-induced silencing of Mi-msp-1 confers resistance to root-knot nematode Meloidogyne incognita in eggplant. Transgenic Res 28:327–340. https://doi.org/10.1007/s11248-019-00126-5
de Almeida EJ, Van Poucke K, Karimi M et al (2004) Dynamic cytoskeleton rearrangements in giant cells and syncytia of nematode-infected roots. Plant J 38:12–26. https://doi.org/10.1111/j.1365-313X.2004.02019.x
de Almeida EJ, de Siqueira KMS, Nascimento DC, do, et al (2016) A cellular outlook of galls induced by root-knot nematodes in the model host Arabidopsis thaliana. Nematoda 3:1–13. https://doi.org/10.4322/nematoda.00616
Desaeger J, Dickson DW, Locascio SJ (2017) Methyl bromide alternatives for control of root-knot nematode (Meloidogyne spp.) in tomato production in Florida. J Nematol 49:140–149. https://doi.org/10.21307/jofnem-2017-058
Djian-Caporalino C, Palloix A, Fazari A et al (2014) Pyramiding, alternating or mixing: comparative performances of deployment strategies of nematode resistance genes to promote plant resistance efficiency and durability. BMC Plant Biol 14:1–13. https://doi.org/10.1186/1471-2229-14-53
Dormatey R, Sun C, Ali K et al (2020) Gene pyramiding for sustainable crop improvement against biotic and abiotic stresses. Agronomy 10:1–20. https://doi.org/10.3390/agronomy10091255
Doyle JJ, Doyle JL (1987) A rapid DNA isolation procedure for small quantities of fresh leaf tissue. Phytochem Bull 9:11–15
Dutta TK, Papolu PK, Banakar P et al (2015) Tomato transgenic plants expressing hairpin construct of a nematode protease gene conferred enhanced resistance to root-knot nematodes. Front Microbiol 6:1–14. https://doi.org/10.3389/fmicb.2015.00260
Dutta TK, Khan MR, Phani V (2019) Plant-parasitic nematode management via biofumigation using brassica and non-brassica plants: current status and future prospects. Curr Plant Biol 17:17–32. https://doi.org/10.1016/j.cpb.2019.02.001
Elling AA (2013) Major emerging problems with minor Meloidogyne species. Phytopathology 103:1092–1102. https://doi.org/10.1094/PHYTO-01-13-0019-RVW
Escobar C, Barcalaa M, Cabrera J, Fenoll C (2015) Overview of root-knot nematodes and giant cells. In: Escobar C, Fenoll C (eds) Advances in botanical research plant nematode interactions: A view on compatible interrelationships. Elsevier, San Diego, CA, pp 1–32. https://doi.org/10.1016/bs.abr.2015.01.001
Grossi-de-Sa MF, Guimarães LM, Batista, JAN, et al (2013) Compositions and methods for modifying gene expression using the promoter of ubiquitin conjugating protein coding gene of soybean plants. Patent US9012720B2.
Hellemans J, Mortier G, De Paepe A, Speleman F, Vandesompele J (2007) qBase relative quantification framework and software for management and automated analysis of real-time quantitative PCR data. Genome Biol 8:1–14. https://doi.org/10.1186/gb-2007-8-2-r19
Ibrahim HMM, Ahmad EM, Martínez-Medina A, Aly MAM (2019) Effective approaches to study the plant-root knot nematode interaction. Plant Physiol Biochem 141:332–342. https://doi.org/10.1016/j.plaphy.2019.06.009
Iqbal MJ, Reddy OUK, El-Zik KM, Pepper AE (2001) A genetic bottleneck in the “evolution under domestication” of upland cotton Gossypium hirsutum L. examined using DNA fingerprinting. Theor Appl Genet 103:547–554. https://doi.org/10.1007/PL00002908
Iqbal S, Fosu-Nyarko J, Jones MGK (2020) Attempt to silence genes of the RNAi pathways of the root-knot nematode, Meloidogyne incognita results in diverse responses including increase and no change in expression of some genes. Front Plant Sci 11:1–13. https://doi.org/10.3389/fpls.2020.00328
ISAAA (2020) Global status of commercialized biotech/GM crops in 2019: Biotech crops drive socio-economic development and sustainable environment in the new frontier, ISAAA Brie. ISAAA, Ithaca, NY
Joshi I, Kumar A, Kohli D et al (2020) Conferring root-knot nematode resistance via host-delivered RNAi-mediated silencing of four Mi-msp genes in Arabidopsis. Plant Sci 298:110592. https://doi.org/10.1016/j.plantsci.2020.110592
Khanal C, McGawley EC, Overstreet C, Stetina SR (2018) The elusive search for reniform nematode resistance in cotton. Phytopathology 108:532–541. https://doi.org/10.1094/PHYTO-09-17-0320-RVW
Krenchinski FH, Albrecht AJP, Salomão Cesco VJ et al (2018) Post-emergent applications of isolated and combined herbicides on corn culture with cp4-epsps and pat genes. Crop Prot 106:156–162. https://doi.org/10.1016/j.cropro.2017.11.016
Kumar A, Kakrana A, Sirohi A et al (2017) Host-delivered RNAi-mediated root-knot nematode resistance in Arabidopsis by targeting splicing factor and integrase genes. J Gen Plant Pathol 83:91–97. https://doi.org/10.1007/s10327-017-0701-3
Kumar A, Jindal SK, Dhaliwal MS et al (2019a) Gene pyramiding for elite tomato genotypes against ToLCV (Begomovirus spp.), late blight (Phytophthora infestans) and RKN (Meloidogyne spp.) for northern India farmers. Physiol Mol Biol Plants 25:1197–1209. https://doi.org/10.1007/s12298-019-00700-5
Kumar P, Khanal S, Da Silva M et al (2019b) Transcriptome analysis of a nematode resistant and susceptible upland cotton line at two critical stages of Meloidogyne incognita infection and development. PLoS ONE 14:1–19. https://doi.org/10.1371/journal.pone.0221328
Kyndt T, Fernandez D, Gheysen G (2014) Plant-parasitic nematode infections in rice: molecular and cellular insights. Annu Rev Phytopathol 52:135–153. https://doi.org/10.1146/annurev-phyto-102313-050111
Li J, Todd TC, Trick HN (2010) Rapid in planta evaluation of root expressed transgenes in chimeric soybean plants. Plant Cell Rep 29:113–123. https://doi.org/10.1007/s00299-009-0803-2
Li F, Fan G, Wang K et al (2014) Genome sequence of the cultivated cotton Gossypium arboreum. Nat Genet 46:567–572. https://doi.org/10.1038/ng.2987
Lourenço-Tessutti IT, Souza Júnior JD, Martins-de-Sá D et al (2015) Knock-down of heat-shock protein 90 and isocitrate lyase gene expression reduced root-knot nematode reproduction. Phytopathology 105:628–637. https://doi.org/10.1094/PHYTO-09-14-0237-R
Mani V, Reddy CS, Lee SK et al (2020) Chitin biosynthesis inhibition of Meloidogyne incognita by RNAi-mediated gene silencing increases resistance to transgenic tobacco plants. Int J Mol Sci 21:1–17. https://doi.org/10.3390/ijms21186626
MAPA (2014) BRS 372 cultivar. In: Ministério da Agric. Pecuária e Abast.—Cultiv. http://sistemas.agricultura.gov.br/snpc/cultivarweb/detalhe_protecao.php?codsr=4800. Accessed 27 May 2021
Mathew R, Opperman CH (2020) Current insights into migratory endoparasitism: deciphering the biology, parasitism mechanisms, and management strategies of key migratory endoparasitic phytonematodes. Plants 9:1–17. https://doi.org/10.3390/plants9060671
McCarter JP, Dautova Mitreva M, Martin J et al (2003) Analysis and functional classification of transcripts from the nematode Meloidogyne incognita. Genome Biol 4:R26. https://doi.org/10.1186/gb-2003-4-4-r26
Niu J, Liu P, Liu Q et al (2016) Msp40 effector of root-knot nematode manipulates plant immunity to facilitate parasitism. Sci Rep 6:1–13. https://doi.org/10.1038/srep19443
Pabinger S, Rödiger S, Kriegner A et al (2014) A survey of tools for the analysis of quantitative PCR (qPCR) data. Biomol Detect Quantif 1:23–33. https://doi.org/10.1016/j.bdq.2014.08.002
Paes de Melo B, Lourenço-Tessutti IT, Morgante CV et al (2020) Soybean embryonic axis transformation: combining biolistic and agrobacterium-mediated protocols to overcome typical complications of in vitro plant regeneration. Front Plant Sci 11:1–14. https://doi.org/10.3389/fpls.2020.01228
Papadopoulou J, Triantaphyllou AC (1982) Sex differentiation in Meloidogyne incognita and anatomical evidence of sex reversal. J Nematol 14:549–566
Papolu PK, Gantasala NP, Kamaraju D et al (2013) Utility of host delivered RNAi of two FMRF amide like peptides, flp-14 and flp-18, for the management of root knot nematode, Meloidogyne incognita. PLoS ONE 8:1–16. https://doi.org/10.1371/journal.pone.0080603
Pérez-Wohlfeil E, Diaz-del-Pino S, Trelles O (2019) Ultra-fast genome comparison for large-scale genomic experiments. Sci Rep 9:1–10. https://doi.org/10.1038/s41598-019-46773-w
Pfaffl MW, Horgan GW, Dempfle L (2002) Relative expression software tool (REST©) for group-wise comparison and statistical analysis of relative expression results in real-time PCR. Nucleic Acids Res 30:e36–e45. https://doi.org/10.1093/nar/30.9.e36
Qi T, Guo J, Peng H et al (2019) Host-induced gene silencing: a powerful strategy to control diseases of wheat and barley. Int J Mol Sci 20:1–15. https://doi.org/10.3390/ijms20010206
R Core Team (2017) R: A language and environment for statistical computing. https://www.r-project.org/. Accessed 24 Jun 2020
Ramalingam J, Raveendra C, Savitha P et al (2020) Gene pyramiding for achieving enhanced resistance to bacterial blight, blast, and sheath blight diseases in rice. Front Plant Sci 11:1–12. https://doi.org/10.3389/fpls.2020.591457
Rech EL, Vianna G, Aragão F (2008) High-efficiency transformation by biolistics of soybean, common bean and cotton transgenic plants. Nat Protoc 3:410–418. https://doi.org/10.1038/nprot.2008.9
Ribeiro TP, Arraes FBM, Lourenço-Tessutti IT et al (2017) Transgenic cotton expressing Cry10Aa toxin confers high resistance to the cotton boll weevil. Plant Biotechnol J 15:997–1009. https://doi.org/10.1111/pbi.12694
Ribeiro TP, Basso MF, Carvalho MH et al (2020) Stability and tissue-specific Cry10Aa overexpression improves cotton resistance to the cotton boll weevil. Biotechnol Res Innov 3:27–41. https://doi.org/10.1016/j.biori.2019.12.003
Rutter WB, Hewezi T, Abubucker S et al (2014) Mining novel effector proteins from the esophageal gland cells of Meloidogyne incognita. Mol Plant-Microbe Interact J 27:965–974. https://doi.org/10.1094/MPMI-03-14-0076-R
Sato K, Kadota Y, Shirasu K (2019) Plant immune responses to parasitic nematodes. Front Plant Sci 10:1–14. https://doi.org/10.3389/fpls.2019.01165
Schmittgen TD, Livak KJ (2008) Analyzing real-time PCR data by the comparative CT method. Nat Protoc 3:1101–1108. https://doi.org/10.1038/nprot.2008.73
Shehryar K, Khan RS, Iqbal A et al (2020) Transgene stacking as effective tool for enhanced disease resistance in plants. Mol Biotechnol 62:1–7. https://doi.org/10.1007/s12033-019-00213-2
Shepherd RL, McCarty JC, Jenkins JN, Parrott WL (1996) Registration of nine cotton germplasm lines resistant to root-knot nematode. Crop Sci 36:820–820. https://doi.org/10.2135/cropsci1996.0011183x003600030071x
Shingles J, Lilley CJ, Atkinson HJ, Urwin PE (2007) Meloidogyne incognita: molecular and biochemical characterisation of a cathepsin L cysteine proteinase and the effect on parasitism following RNAi. Exp Parasitol 115:114–120. https://doi.org/10.1016/j.exppara.2006.07.008
Siddiqui HA, Asif M, Asad S et al (2019) Development and evaluation of double gene transgenic cotton lines expressing Cry toxins for protection against chewing insect pests. Sci Rep 9:1–7. https://doi.org/10.1038/s41598-019-48188-z
Singh R, Phulera S (2015) Plant parasitic nematodes: the hidden enemies of farmers. In: Yadav S, Singh R (eds) Environmental issues for socio-ecological development, 1st edn. Excel India Publishers, New Delhi, pp 68–81. https://doi.org/10.13140/RG.2.1.2994.7763
Sönnichsen B, Koski LB, Walsh A et al (2005) Full-genome RNAi profiling of early embryogenesis in Caenorhabditis elegans. Nature 434:462–469. https://doi.org/10.1038/nature03353
de Souza Júnior JDA, Ramos Coelho R, Tristan Lourenço I et al (2013) Knocking-down Meloidogyne incognita proteases by plant-delivered dsRNA has negative pleiotropic effect on nematode vigor. PLoS ONE 8:1–17. https://doi.org/10.1371/journal.pone.0085364
Suassuna ND, Scoz LB, Giband M (2016) Melhoramento genético do algodoeiro para resistência aos nematoides: seleção assistida por marcadores moleculares. In: Galbieri R, Bellot JL (eds) Nematoides fitoparasitas do algodoeiro nos cerrados brasileiros: biologia e medidas de controle. Instituto Mato-grossense do Algodão, Cuiabá, pp 243–256
Tamilarasan S, Rajam MV (2013) Engineering crop plants for nematode resistance through host-derived RNA interference. Cell Dev Biol 2:2–7. https://doi.org/10.4172/2168-9296.1000114
Tangtrakulwanich K, Reddy GVP (2014) Development of insect resistance to plant biopesticides: an overview. In: Singh D (ed) Advances in plant biopesticides. Springer, New Delhi, pp 47–62. https://doi.org/10.1007/978-81-322-2006-0_4
Thompson CJ, Movva NR, Tizard R et al (1987) Characterization of the herbicide-resistance gene bar from Streptomyces hygroscopicus. EMBO J 6:2519–2523
Tort J, Brindley PJ, Knox D et al (1999) Proteinases and associated genes of parasitic helminths. Adv Parasitol 43:161–266. https://doi.org/10.1016/S0065-308X(08)60243-2
Triantaphyllou AC (1973) Environmental sex differentiation of nematodes in relation to pest management. Annu Rev Phytopathol 11:441–462. https://doi.org/10.1146/annurev.py.11.090173.002301
Vieira P, Danchin EGJ, Neveu C et al (2011) The plant apoplasm is an important recipient compartment for nematode secreted proteins. J Exp Bot 62:1241–1253. https://doi.org/10.1093/jxb/erq352
Vieira P, Eves-Van Den Akker S, Verma R et al (2015) The Pratylenchus penetrans transcriptome as a source for the development of alternative control strategies: mining for putative genes involved in parasitism and evaluation of in planta RNAi. PLoS ONE 10:1–25. https://doi.org/10.1371/journal.pone.0144674
Wadsworth WG, Riddle DL (1989) Developmental regulation of energy metabolism in Caenorhabditis elegans. Dev Biol 132:167–173. https://doi.org/10.1016/0012-1606(89)90214-5
Walawage SL, Britton MT, Leslie CA et al (2013) Stacking resistance to crown gall and nematodes in walnut rootstocks. BMC Genomics 14:1–13. https://doi.org/10.1186/1471-2164-14-668
Wang M, Wu L, Mei Y et al (2020) Host-induced gene silencing of multiple genes of Fusarium graminearum enhances resistance to Fusarium head blight in wheat. Plant Biotechnol J 18:2373–2375. https://doi.org/10.1111/pbi.13401
Wubben MJ, Thyssen GN, Callahan FE et al (2019) A novel variant of Gh_D02G0276 is required for root-knot nematode resistance on chromosome 14 (D02) in Upland cotton. Theor Appl Genet 132:1425–1434. https://doi.org/10.1007/s00122-019-03289-1
Yadav BC, Veluthambi K, Subramaniam K (2006) Host-generated double stranded RNA induces RNAi in plant-parasitic nematodes and protects the host from infection. Mol Biochem Parasitol 148:219–222. https://doi.org/10.1016/j.molbiopara.2006.03.013
Yang X, Li F, Liu C et al (2012) Analysis of the copy number of exogenous genes in transgenic cotton using real-time quantitative PCR and the 2-△△CT method. African J Biotechnol 23:6226–6233. https://doi.org/10.5897/AJB11.4117
Zhang J, Hong Y (2013) Investigating transgene integration and organization in cotton (Gossypium hirsutum L.) genome. In: Zhang B (ed) Methods in molecular biology, 1st edn. Humana Press, Greenville, pp 95–107. https://doi.org/10.1007/978-1-4939-8952-2_10
Acknowledgements
The authors gratefully acknowledge the support of EMBRAPA, UCB, CNPq, CAPES, INCT PlantStress Biotech, and FAPDF. We thank the Mato-Grossense Cotton Institute (IMAmt) for its partnership and support in the execution of this project. Authors also thank Josue Inácio Lemos and Marcelo Broillo Paganella for technical support with plant transformation and plant management in the greenhouse.
Funding
PLRS thanks to the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) for the master’s research fellowship and JPAS thanks to the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) for the undergraduation research fellowship. This work was supported by grants from EMBRAPA, UCB, CAPES, INCT-CNPq, FAP-DF, and IMAmt.
Author information
Authors and Affiliations
Corresponding author
Ethics declarations
Conflict of interest
The authors have no conflicts of interest to declare that are relevant to the content of this manuscript. The authors have no relevant financial or non-financial interests to disclose.
Ethics approval and consent to participate
Not applicable.
Consent for publication
Not applicable.
Additional information
Communicated by Dorothea Bartels.
Publisher's Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Supplementary Information
Below is the link to the electronic supplementary material.
425_2021_3776_MOESM1_ESM.tiff
Fig. S1 Schematic representation of the dsMinc1 expression cassette (10,781 bp) used for cotton transformation. The binary vector pCambia3300 was used as a backbone for the RNAi construct, containing in tandem fragments of sense and antisense cDNA sequences of M. incognita genes Mi-cpl, Mi-icl, and Mi-sf under the control of pUceS8.3 promoter and Nos terminator (Nos-t). Selection markers based on herbicide-resistance comprise the acetohydroxyacid synthase (ahas) gene, controlled by its promoter and terminator (ahas-t), and bialaphos resistance gene (bar), controlled by the 35S RNA double promoter of cauliflower mosaic virus (p35S 2x) and Nos-t. RB, right border; LB, left border (TIFF 185 KB)
425_2021_3776_MOESM2_ESM.tif
Fig. S2 Molecular characterization of T0 independent events (a) and in their respective T4-dsMinc1 cotton plants selected by PCR: amplification of PDK fragments (279 bp). b, c, d, and e dsMinc1-10 lines; f, g, h, and i dsMinc1-20 lines; j, k, and l dsMinc1-64 lines. Lanes with numbers: DNA samples from different T4 cotton events. WT used as a negative control. (-) water used as a negative control for the PCR reaction. (+): positive control - dsMinc1 expression cassette; X, empty well; M, ladder 1kb Plus (Invitrogen). In red, PCR-positive plants; in black, PCR-negative plants (TIF 642 KB)
425_2021_3776_MOESM3_ESM.tif
Fig. S3 Relative expression of Mi-cpl, Mi-icl, and Mi-sf genes in M. incognita from galls in T4-GM cotton events (dsMinc1-10, dsMinc1-20, and dsMinc1-64) and WT plants. Relative expression of a Mi-cpl, b Mi-icl and c Mi-sf genes. Elongation factor (ef1) and actin (act) were used as reference genes for M. incognita. The expression of Mi-cpl, Mi-icl and Mi-sf was quantified by the 2-ΔCt method. Error bars indicate the standard errors of the mean (TIF 875 KB)
Rights and permissions
About this article
Cite this article
Lisei-de-Sá, M.E., Rodrigues-Silva, P.L., Morgante, C.V. et al. Pyramiding dsRNAs increases phytonematode tolerance in cotton plants. Planta 254, 121 (2021). https://doi.org/10.1007/s00425-021-03776-0
Received:
Accepted:
Published:
DOI: https://doi.org/10.1007/s00425-021-03776-0