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Figure 10. Virus induced gene silencing of X2 and X7 abolishes geminiviral accumulation but does not affect infection by RNA viruses. TRV X2 X7 TRV X2 X7 Importance of vesicle trafficking in the establishment of a geminiviral infection Cana-Quijada P, Rosas-Díaz T, Lozano-Durán R and Bejarano ER. Instituto de Hortofruticultura Subtropical y Mediterránea “La Mayora” (IHSM-UMA-CSIC), Área de Genética, Dpto. Biología Celular, Genética y Fisiología. Universidad de Málaga. Facultad de Ciencias. Campus de Teatinos. Málaga. email: edu_r[email protected] INTRODUCTION Geminiviruses are a large family of insect-transmitted plant viruses with circular, single-stranded (ss) DNA genomes packaged within geminated particles which infect a wide range of plants causing devastating crop diseases. From among these diseases, Tomato yellow leaf curl disease (TYLCD), is one of the most important threats to tomato crops worldwide. One of the causal agents of TYLCD is Tomato yellow leaf curl Sardinian virus (TYLCSV), a member of the genus Begomovirus belonging to the family Geminiviridae. TYLCSV has a monopartite genome, which encodes six proteins and contains an intergenic region (IR) comprising the origin of replication and viral promoters. Due to the few proteins encoded by the viral genome, they rely heavily on host cellular machineries and interact with a wide range of plant proteins to complete all processes required for infection, such as viral replication, movement, and suppression or evasion of plant defence mechanisms. While cell-to-cell movement has been described to occur through plasmodesmata (Zhou et al., 2011), the way in which geminiviruses move inside the host cells is yet unknown. Here we describe how vesicle trafficking is essential for viral infection. Figure 2. The begomovirus life cycle. (Adapted from Hanley-Bowdoin et al., 2013) Figure 1. Geographical distribution (A) and symptoms of TYLCD (B). (taken from Navas-Castillo et al., 2011) A B 35S IR IR GFP NOS LB RB v -TYLCSV +TYLCSV A B C Figure 3. Nicotiana benthamiana 2IR GFP plants. A) Construct of 2IR GFP plants. B) Episomal replicon formation in 2IR plants infected with TYLCSV. C) GFP expression in 2IR GFP plants under UV light at 15 dpi . Rep protein GFP protein Control: TRV+TYLCSV TRV-GOI+TYLCSV TRV-GOI silencing TYLCSV infection A B GFP expression and phenotype observation C 7 - 15 dpi Figure 5. Strategy used to identify genes required for TYLCSV infection in Nicotiana benthamiana 2IR GFP plants. A) Silencing of the gene of interest (GOI) through infiltration with Agrobacterium containing a TRVGOI construct. B) Immediately afterwards, an infection is carried out through injection with Agrobacterium containing TYLCSV. C) Plants are observed under UV light to see the evolution of GFP expression from days 7 to 15 post infection. GOI Figure 4. A) Genomic organization of TRV vector. RNA 2 contains a multiple cloning site (MCS) where the genes of interest (GOI) have been cloned and, therefore, will be silenced together with the viral mRNA in N.benthamiana plants. B) Coinfection with TRV does not affect TYLCSV infection. Silencing of viral mRNA and endogenous GOI TYLCSV TYLCSV + TRV AB X2 X7 W5 Z4 Z7 Z8 Y3 Y5 Figure 6. Selected genes for TRV silencing and their place in the different pathways of vesicular transport. Figure 7. GFP expression in the last 3 leaves of 2IR plants silenced with the different GOIs and infected with TYLCSV at 15dpi. MATERIAL AND METHODOLOGY Acknowledgements: This research was supported by a grant from the Spanish Ministerio de Ciencia y Tecnología (AGL2013-48913-C2-2-R). REFERENCES (1) Zhou Y. Et al. (2011) Histone H3 Interacts and Colocalizes with the Nuclear Shuttle Protein and the Movement Protein of a Geminivirus. Journal of Virology. 85 (22) 11821–11832. (2) Morilla G. et al. (2006) A Versatile Transreplication-Based System To Identify Cellular Proteins Involved in Geminivirus Replication. Journal of Virology. 80 (7) 3624-3633. (3)Navas-Castillo et al. (2011) Emerging Virus Diseases Transmitted by Whiteflies. Annual Review of Phytopathology. 49:219–48 (4) Hanley-Bowdoin, L et al. (2013). Geminiviruses: masters at redirecting and reprogramming plant processes. Nature reviews. Microbiology 11, 777-788. (5) Hsu, V.W., Lee, S.Y., Yang, J.S., 2009. The evolving understanding of COPI vesicle formation. Nature reviews. Molecular cell biology 10, 360-364. (6) Lozano-Durán, R., Rosas-Diaz, T., Luna, A.P., Bejarano, E.R., 2011b. Identification of host genes involved in geminivirus infection using a reverse genetics approach. PLoS One 6, e22383. Figure 8. Evolution of GFP expression levels in 2IR plants silenced with the different GOIs and infected with TYLCSV during days 7-15 post-infection. Silencing of genes X2, X7, Z7 and Z8, produced a clear effect over TYLCSV infection, dropping the levels of GFP expression and viral DNA to 0or almost 0. For plants which had genes X2 and X7 silenced different infection assays were carried. These plants were separately infected with TYLCSV (a begomovirus), BCTV (a curtovirus) and two different RNA viruses: PVX and TMV. Both geminiviruses presented almost null levels of DNA meanwhile both RNA viruses were not affected by the silencing. This data unveils a role of the COPIdependent retrograde transport as an essential and specific pathway for geminivirus infection. For both Z7 and Z8, the measurement of relative amounts of TYLCSV DNA confirmed the preliminary results which seem to dramatically affect infection. The fact that these two genes and not Z4 and W5, involved in other parts of the membrane transport and endosomes pathway, affect infection remains a subject still to elucidate. Genes involved in anterograde transport did not notably affect infection, Y5 did promote a slightly higher accumulation of viral DNA. RESULTS AND DISCUSSION In our laboratory, transgenic Nicotiana benthamiana plants containing a green fluorescent protein (GFP) expression cassette flanked by two direct repeats of the intergenic region of TYLCSV have been constructed (2IR plants) (Morilla et al., 2006). These plants have been used together with virus induced gene silencing (VIGS) based on a TRV vector, in an effort to identify host genes involved in the infection process using a reverse genetics approach. As a result, two genes involved in vesicular trafficking were identified such as: X2 and X7. A set of genes involved in this process were later assayed in order to see their effect over infection (genes Z4, Z7, Z8, Y3, Y5 and W5). The identification of the host proteins involved in viral infection will be an important step towards the understanding of the mechanisms underlying this process. RNA 2 Lb 35S CP TRb MCS 16KMP Lb 35S RdRp TRb RNA 1 TYLCSV TYLCSV + TRV Y3 X2 Z8 W5 0 0,5 1 1,5 2 2,5 3 7 9 11 13 15 GFP levels dpi MEMBRANE TRANSPORT AND ENDOSOMES TRV TriskV1 TriskV2 AP1 SYT1 0 0,5 1 1,5 2 2,5 3 7 9 11 13 15 GFP levels dpi ANTEROGRADE TRANSPORT TRV Sec24 Sar1 Y3 Y5 Z7 Z8 Z4 W5 0 0,5 1 1,5 2 2,5 3 7 9 11 13 15 GFP levels dpi RETROGRADE TRANSPORT TRV ARF1 δCOP X2 X7 Figure 9. TYLCSV accumulation in plants with the different silenced genes ns * ns ns **** **** 0,0 1,0 2,0 3,0 4,0 AP1 TriskV1 TriskV2 Sar1 Sec24 Syt1 COP ARF TRV TYLCSV DNA TYLCSV quantitation W5 Z4 Z8 Z7 Y3 Y5 TRV X2 X7 nd **** TRV X2 X7
