Agroinoculation of Beet necrotic yellow vein virus cDNA clones results in plant systemic infection and efficient Polymyxa betae transmission

Abstract

Agroinoculation is a quick and easy method for the infection of plants with viruses. This method involves the infiltration of tissue with a suspension of Agrobacterium tumefaciens carrying binary plasmids harbouring full-length cDNA copies of viral genome components. When transferred into host cells, transcription of the cDNA produces RNA copies of the viral genome that initiate infection. We produced full-length cDNA corresponding to Beet necrotic yellow vein virus (BNYVV) RNAs and derived replicon vectors expressing viral and fluorescent proteins in pJL89 binary plasmid under the control of the Cauliflower mosaic virus 35S promoter. We infected Nicotiana benthamiana and Beta macrocarpa plants with BNYVV by leaf agroinfiltration of combinations of agrobacteria carrying full-length cDNA clones of BNYVV RNAs. We validated the ability of agroclones to reproduce a complete viral cycle, from replication to cell-to-cell and systemic movement and, finally, plant-to-plant transmission by its plasmodiophorid vector. We also showed successful root agroinfection of B. vulgaris, a new tool for the assay of resistance to rhizomania, the sugar beet disease caused by BNYVV.

Figure 1

(a) Beet necrotic yellow vein virus (BNYVV) constructs built and used in this study. Each clone has the same pJL89‐derived backbone. Double StuI/SmaI (*) restriction digestion allows the insertion of blunt‐end polymerase chain reaction (PCR) fragments between the double 35S promoter (2‐35S) and the hepatitis delta virus ribozyme (Rz) followed by a Nos terminator. LB and RB represent the left and right borders of the T‐DNA. Mtr, methyltransferase; Hel, helicase; Pro, protease; Pol, RNA polymerase; RT, readthrough; RTD, readthrough domain; star, suppressible UAG stop codon; CRP, cysteine‐rich protein; GFP, green fluorescent protein; Ha, haemagglutinin tag. (b) Schematic representation of pB15 and pB15–SphI–MluI clones used for the production of AgroBN‐1. Broken lines represent segments between restriction sites SphI and MluI removed from BNYVV RNA‐1 clone (pB15) in order to facilitate cloning in the pJL89‐SphI plasmid.

Figure 2

Symptom expression, particle detection and reporter gene expression obtained by agroinfiltration of Agrobacterium tumefaciens cells carrying AgroBN‐1 to AgroBN‐5 clones (a, b, c, d and f) or AgroBN‐1 to AgroBN‐4 and AgroRep5GFP clones (e and g). (a) Local symptoms in agroinfiltrated leaf of Nicotiana benthamiana at 7 days post‐inoculation (dpi). (b) Symptoms in systemically infected leaf of N. benthamiana at 14 dpi. (c) Local chlorotic spots in leaf of Beta macrocarpa agroinfiltrated with AgroBN‐1 to AgroBN‐5 clones (left) and symptomless leaf of B. macrocarpa agroinfiltrated with empty pJL89 binary vector (right) at 7 dpi. (d) Systemic symptom development in B. macrocarpa: nonagroinfected plant on the left, infected plant in the middle, showing some distorted leaves (14 dpi), and infected plant with totally distorted leaves on the right (30 dpi). (e) Agroclone‐mediated green fluorescent protein (GFP) expression in N. benthamiana cells. (f) Beet necrotic yellow vein virus (BNYVV) particles observed in B. macrocarpa roots at transmission electron microscopy (TEM) by immunosorbent electron microscopy (ISEM) (14 dpi). Bar represents 200 nm. (g) Agroclone‐mediated GFP expression in agroinfected roots of B. macrocarpa, visualized by confocal microscopy (14 dpi).

Figure 3

Northern (a) and Western (b) blot analysis of Nicotiana benthamiana agroinoculated leaves: 1, AgroBN‐1 + AgroBN‐2 + AgroRep5p25Ha; 2, AgroBN‐1 + AgroBN‐2 + AgroRep3p29Ha; 3, AgroBN‐1 + AgroBN‐2 + AgroRep3GFP; 4, AgroBN‐1 + AgroBN‐2 + AgroRep5GFP. The equal loading of the Northern blot was visualized by ethidium bromide (EtBr) staining. Different RNAs were detected using complementary riboprobes. Immunodetection of virally expressed protein was performed on equally loaded gel as shown with membrane staining (MS).