Development of T3SS Mutants (hrpB- and hrcV-) of Ralstonia solanacearum, Evaluation of Virulence Attenuation in Brinjal and Tomato-A Pre-requisite to Validate T3Es of R. solanacearum

Abstract

Ralstonia solanacearum is an important plant pathogen which infects a large number of agriculturally important crops. The Type Three Secretion System (T3SS) plays a major role in its pathogenicity by secreting type III effectors (T3Es) which overthrow the host defence mechanism. The secretion of T3Es is transcriptionally regulated by hrpB and its secretion is dependent on the pili formed by hrcV gene. In this study, two T3SS mutants of R. solanacearum strain Rs-09-161 viz. Rs-HrpB^- and Rs-HrcV^- were developed through insertional mutagenesis. The method of development of insertional mutant is quite simple and reliable. The plasmid integrates through homologous recombination and in vitro studies have proved that the integration was stable for several generations. The mutants are non-pathogenic on its highly susceptible hosts, brinjal and tomato inoculated by soil drench method and by petiole inoculation directly into the vascular system. Further it was observed that the colonisation ability of the mutants was also highly reduced in the susceptible host. These mutants will be useful in validating putative T3E through translocation studies.

Keywords: Bacterial wilt; Mutant; Ralstonia solanacearum; Type three secretion system.

Fig. 1

Twitching motility displayed by the R. solanacearum strain Rs-09-161 observed at 10× magnification after 18–20 h. Each raft of motile cells forms an individual colony which can be easily visualised by naked eyes after 48 h

Fig. 2

Cloning of internal fragment of HrpB gene in R. solanacearum a amplification of internal fragment of HrpB gene. Lane 1: Ladder (100 bp–5 Kb, GeneRuler Express DNA Ladder), lane 2: 582 bp. b Colony PCR for the confirmation of HrpB insert in E.coli colonies. Lane 1: Ladder (100 bp–5 Kb, GeneRuler Express DNA Ladder), Lane 2–7: 582 bp. c Restriction digestion of plasmid hrpBpTZ57R/T which contains the internal fragment of HrpB gene cloned into it. Lane 1 contains the 100 bp–5 Kb ladder (GeneRuler Express DNA Ladder); lane 2: Rsa I digest with 3 bands at 2333, 1066 and 72 bp; lane 3: Bam HI/Eco RI double digest with 3 bands of 2847, 403 and 221 bp; lane 4: Sac I/Not I double digest with 2 bands of 3361 and 110 bp; lane 5: Eco RI digest with 2 bands at 3250 and 221 bp; lane 7: 100 bp–1 Kb ladder; lane 7: uncut plasmid

Fig. 3

Mutagenesis of hrcV gene in R. solanacearum Rs-09-161. a Colony PCR for the confirmation of HrcV insert in E. coli colonies. Lane 1: Ladder (100 bp–5 Kb, GeneRuler Express DNA Ladder), Lane 2–4: 763 bp. b Restriction digestion of plasmid hrcVpTZ57R/T which contains the internal fragment of HrcV gene cloned into it. Lane 1: 100 bp–5 Kb (GeneRuler Express DNA Ladder) ladder; lane 2: uncut plasmid hrcVpTZ57R/T; lane 3: Bam HI/Eco RI double digest with 2 bands of 2847, 802 bp; lane 4: Rsa I digest with 4 bands at 1987, 1066, 483 and 113 bp. c Confirmation of insertional mutagenesis in R. solanacearum Rs-09-161. Lane 1: 100 bp–5 kb (GeneRuler Express DNA Ladder) ladder; lane 2: 1628 bp obtained by disruption of hrpB gene in Rs-hrpB⁻; lane 3: 1624 bp obtained by disruption of hrcV gene in Rs-hrcV⁻

Fig. 4

Virulence assay in brinjal inoculated by soil drench with wild type R. solanacearum Rs-09-161 and T3SS mutants Rs-hrpB⁻ and Rs-hrcV⁻. Wilt % is mean of 2 experiments conducted separately with 2 and 3 replications in experiment 1 and experiment 2 respectively. Each replication had 5 plants. The plants were observed for 21 days post inoculation

Fig. 5

Virulence assay in brinjal inoculated on cut petiole with wild type R. solanacearum Rs-09-161 and T3SS mutants Rs-hrpB⁻ and Rs-hrcV⁻. Wilt % is a mean of 2 experiments conducted separately with 2 and 3 replications in experiment 1 and experiment 2 respectively. Each replication had 5 plants. The plants were observed for 21 days post inoculation

Fig. 6

In planta assays using R. solanacearum strains Rs-09-161, Rs-hrpB⁻ and Rs-hrcV⁻. a 100% wilt caused by wild type Rs-09-161 on its susceptible hosts brinjal cultivar Agassaim and tomato variety Pusa ruby. b Tomato seedlings (Pusa ruby) inoculated on the petiole with 2000 cells of R. solanacearum strains Rs-hrpB⁻, Rs-hrcV⁻ and Rs-09-161; Rs-09-161 inoculated plants show complete wilt whereas Rs-hrpB⁻ inoculated do not display any wilt symptom. c Brinjal cultivar Agassaim inoculated with 10 ml inoculum of 0.1 OD_600nm of R. solanacearum strains Rs-hrpB⁻, Rs-hrcV⁻ and Rs-09-161 by drench inoculation; Rs-09-161 displays wilt symptoms whereas no wilt is observed with plants inoculated with Rs-hrpB⁻ and Rs-hrcV⁻

Fig. 7

Virulence assay in tomato inoculated by soil drench with wild type R. solanacearum Rs-09-161 and T3SS mutants Rs-hrpB⁻ and Rs-hrcV⁻. Wilt % is mean of 2 experiments conducted separately with 3 replications each. Each replication had 5 plants. The plants were observed for 21 days post inoculation

Fig. 8

Virulence assay in tomato inoculated on cut petiole with wild type R. solanacearum Rs-09-161 and T3SS mutants Rs-hrpB⁻ and Rs-hrcV⁻. Wilt % is mean of 3 experiments conducted separately with 2 replications in experiment 1 and 3 replications in experiment 2 and 3 respectively. Each replication had 5 plants. The plants were observed for 21 days post inoculation