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Interfamily transfer of Bs2 from pepper to cassava (Manihot esculenta Crantz)

Tropical Plant Pathology volume 44, pages 225–237 (2019)Cite this article

Abstract

Cassava (Manihot esculenta Crantz) is the principal source of calories among root and tuber crops in tropical countries. Cassava bacterial blight (CBB) caused by Xanthomonas axonopodis pv. manihotis (Xam) is the most prevalent bacterial disease in cassava. Genome and mutagenesis analysis of Xam strains has led to the identification of an effector similar to avrBs2 from Xanthomonas euvesicatoria among a core set effectors contributing to Xam virulence. Previous studies have demonstrated that transgenic tomato plants expressing Bs2, the AvrBs2 cognate resistance protein from pepper, are resistant to Xanthomonas euvesicatoria. According to avrBs2 wide distribution and important contribution to Xam virulence, we aimed at overexpressing Bs2 in cassava plants as a strategy to control CBB. The susceptible cultivar 60444 was transformed with the Bs2 gene from pepper and transgenic cassava plants that functionally express Bs2 were regenerated. Our results show that overexpression of Bs2 in a highly susceptible cultivar leads to reactive oxygen species production. However, the overexpression of Bs2 neither leads to an HR in cassava nor reduces Xam growth on in vitro plants. These results suggest that BS2 activates defense-signaling pathways in cassava such as ROS production, although this is not sufficient to restrict Xam growth.

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Acknowledgements

The authors would like to acknowledge Emily McCallum, Hervé Vanderschuren and Wilhelm Gruissem (ETH Zürich, Switzerland) for comments and support during PADT’s internship in their laboratory, and Camilo Dorado for his support on the statistical analysis. The Bs2 gene was kindly provided by Brian Staskawicz (University of California, Berkeley, USA). This research was supported by a National Science Foundation/BREAD (Basic Research to Enable Agricultural Development) grant (Award 0965418). PADT was supported with a scholarship for graduate students from Universidad Nacional de Colombia. The authors have no conflict of interest to declare.

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  1. Paula A. Díaz-Tatis

    Present address: Grupo de Ciencias Biológicas y Químicas, Departamento de Biología, Universidad Antonio Nariño, Cra1 #47a15, Bogotá, Colombia

  2. Juan C. Ochoa

    Present address: Institute of Plant Genetics, Department of Integrative Biology, Polish Academy of Sciences, Strzeszynska, 34 60-479, Poznan, Poland

Authors and Affiliations

  1. Manihot Biotec, Departamento de Biología, Universidad Nacional de Colombia, Cra30 #45-03, Bogotá D.C., Colombia

    Paula A. Díaz-Tatis, Juan C. Ochoa, Lina García & Camilo E. López

  2. Transformation Platform, Centro Internacional de Agricultura Tropical (CIAT), Km17 Cali-Palmira, Palmira, Colombia

    Paul Chavarriaga

  3. Laboratorio de Interacciones Moleculares de Microorganismos Agrícolas (LIMMA), Departamento de Ciencias Biológicas, Universidad de los Andes, Cra1 #18a-12, Bogotá D.C., Colombia

    Adriana J. Bernal

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Supplementary Table S1

- List of primers used in this study. (XLSX 55 kb)

Supplementary Table S2

- Result of BLASTp analysis against the cassava proteome (v. 6.1) using the Bs2 sequence from pepper as a query. (XLSX 64 kb)

Supplementary Figure S1

- Transient over-expression of Bs2 and Bs2(D475V) assays in N. benthamiana and N. tabacum. aN. benthamiana leaves were infiltrated with A. tumefaciens strain GV3101 (OD600nm = 0,3) transformed with the binary vectors pBAV139 containing 35S::Bs2, 35S::Bs2(D475V),35S::Pto(L205D) and an empty vector (EV). b Destained leaf shown in a. cN. tabacum leaves were infiltrated with A. tumefaciens strain GV3101 (OD600nm = 0,3) transformed with the binary vectors pBAV139 containing 35S::Bs2, 35S::Bs2(D475V),35S::Pto(L205D) and an EV. d Destained leaf shown in c. (PNG 2143 kb)

High Resolution Image (TIF 12572 kb)

Supplementary Figure S2

- Transient overexpression of Bs2 and Bs2(D475V) assays in cassava leaves. a Cassava leaves (cultivar SG10735) were infiltrated with A. tumefaciens strain AGL1 (OD600nm = 0,3) transformed with the binary vectors pBAV139 containing 35S::Bs2, 35S::Bs2(D475V) and an empty vector (EV). b Destained leaf shown in a. (PNG 615 kb)

High Resolution Image (TIF 3706 kb)

Supplementary Figure S3

- PCR analysis of Bs2 cassava transgenic plants. Genomic DNA was isolated from fresh leaves and a PCR was performed using specific primers for Bs2, Bs2_F and Bs2_R in Suppl. Table S1 (a). PP2A was used as an endogenous control; primers used are listed in Suppl. Table S1 (b). L.1-L.11, genomic DNA from the different transgenic plants obtained; M, 1 Kb plus DNA ladder; −, negative control. (PNG 382 kb)

High Resolution Image (TIF 29564 kb)

Supplementary Figure S4

- Southern-blot analysis of Bs2 cassava transgenic plants. Genomic DNA was isolated from fresh leaves and a southern Blot was performed using hptII as a probe. L.1-L.11, genomic DNA from the different transgenic plants obtained; WT, genomic DNA from wild-type (non-transformed) plants used as a negative control. (PNG 679 kb)

High Resolution Image (TIF 14573 kb)

Supplementary Figure S5

- Differences in the phenotype of in vitro wild-type (non-transformed; WT) plants (a), empty vector (EV) transgenic plants (b) and Bs2 transgenic plants (c). The pictures were taken to one-month-old in vitro plants. (PNG 2115 kb)

High Resolution Image (TIF 15662 kb)

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Díaz-Tatis, P.A., Ochoa, J.C., García, L. et al. Interfamily transfer of Bs2 from pepper to cassava (Manihot esculenta Crantz). Trop. plant pathol. 44, 225–237 (2019). https://doi.org/10.1007/s40858-019-00279-y

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Keywords

  • Cassava bacterial blight
  • Heterologous system
  • Nucleotide-binding leucine-rich repeats
  • Plant immunity
  • R gene interfamily transfer
  • Transgenic cassava

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