Katalin Nemes

Alanine Scanning of Cucumber Mosaic Virus (CMV) 2B Protein Identifies Different Positions for Cell-To-Cell Movement and Gene Silencing Suppressor Activity

The multifunctional 2b protein of CMV has a role in the long distance and local movement of the virus, in symptom formation, in evasion of defense mediated by salicylic acid as well as in suppression of RNA silencing. The role of conserved amino acid sequence domains were analyzed previously in the protein function, but comprehensive analysis of this protein was not carried out until recently. We have analyzed all over the 2b protein by alanine scanning mutagenesis changing three consecutive amino acids (aa) to alanine. We have identified eight aa triplets as key determinants of the 2b protein function in virus infection. Four of them (KKQ/22-24/AAA, QNR/31-33/AAA, RER/34-36/AAA, SPS/40-42/AAA) overlap with previously determined regions indispensable in gene silencing suppressor function. We have identified two additional triplets necessary for the suppressor function of the 2b protein (LPF/55-57/AAA, NVE/10-12/AAA), and two other positions were required for cell-to-cell movement of the virus (MEL/1-3/AAA, RHV/70-72/AAA), which are not essential for suppressor activity.

The C-terminal domain of the 2b protein of Cucumber mosaic virus is stabilized by divalent metal ion coordination.

The main function of the 2b protein of Cucumber mosaic virus (CMV) is binding permanently the double stranded siRNA molecules in the suppression process of post-transcriptional gene silencing (PTGS). The crystal structure of the homologue Tomato aspermy virus (TAV) 2b protein is known, but without the C-terminal domain. The biologically active form is a tetramer: four 2b protein molecules and two siRNA duplexes. Regarding the complete 2b protein structure, we performed a molecular dynamics (MD) simulation of the whole siRNA-2b ribonucleoprotein complex. Unfortunately, the C-terminal domain is proved to be partially unstructured. Multiple sequence alignment showed a well conserved motif between residues 94 and 105. The negatively charged residues of the C-terminal domain are supposed to take part in coordination of a divalent metal ion and stabilize the three-dimensional structure of the C-terminal domain. MD simulations were performed on the detached C-terminal domains (aa 65-110). 0.15 M MgC₂, CaCl₂, FeCl₂ and ZnCl₂ salt concentrations were used in the screening simulations. Among the tested divalent metal ions Mg²⁺ proved to be very successful because Asp95, Asp96 and Asp98 forms a quasi-permanent Mg²⁺ binding site. However the control computations have resulted in any (at least) divalent metal ion remains in the binding site after replacement of the bound Mg²⁺ ion. A quadruple mutation (Rs2DDTD/95-98/AAAA) was introduced into the position of the putative divalent metal ion binding site to analyze the biological relevance of molecular modeling derived hypothesis. The plant inoculation experiments proved that the movement of the mutant virus is slower and the symptoms are milder comparing to the wild type virus. These results demonstrate that the quadruple mutation weakens the stability of the 2b protein tetramer-siRNA ribonucleoprotein complex.

INVESTIGATION ON TOMATO SPOTTED WILT VIRUS INFECTING PEPPER PLANTS IN HUNGARY

In Hungary resurgence of Tomato spotted wilt virus (TSWV) frequently causes heavy crop losses in pepper production since the mid nineties. Management of TSWV control was first directed against the thrips (using different insecticides or plastic traps), and against weeds as host plants of the virus and the thrips. Later on Tsw resistance gene was introduced from Capsicum chinense PI 152225 and PI 159236 into different types of pepper. In 2010 and 2011 sporadically, but in 2012 more frequently a resistance breaking (RB) strain of TSWV on resistant pepper cultivars was observed in the Szentes region (South-East Hungary). The presence of a new resistance breaking strain was demonstrated by virological (test-plant, serological and RT-PCR) methods. Previously, the non-structural protein (NSs) encoded by small RNA (S RNA) of TSWV was verified as the avirulence factor for Tsw resistance, therefore we analyzed the S RNA of the Hungarian RB and wild type (WT) isolates and compared to previously analyzed TSWV strains with RB properties from different geographical origins. Phylogenetic analysis demonstrated that the different RB strains had the closest relationship with the local WT isolates and there was no conserved mutation present in all the NSs genes of RB isolates from different geographical origins. According to these results, it is concluded that the RB isolates evolved separately in geographic point of view and according to the RB mechanism. In order to find new genetic sources of resistance in Capsicum species 89 lines from Capsicum annuum, C. chinense, C. frutescens, C. chacoense, C. baccatum var. baccatum, C. baccatum var. pendulum and C. praetermissum were tested with the Hungarian TSWV-RB isolate.