An Amino Acid Substitution at Position 740 in σ ⁷⁰ of Ralstonia solanacearum Strain OE1-1 Affects Its In Planta Growth

Abstract

Growth of Ralstonia solanacearum strain OE1-1 in roots after invasion is required for virulence. An Arg740Cys substitution in 70 of OE1-1 resulted in loss of in planta growth and virulence. The negative dominance of mutant 70 over the wild-type protein suggested that the amino acid substitution may affect the in planta growth of OE1-1, leading to a lack of virulence.Ralstonia solanacearum strain OE1-1 shows virulence on tobacco, as well as tomato and eggplant, plants (17). OE1-1 possesses hrp genes, similar to R. solanacearum strain GMI1000, which is virulent on tomato plants and elicits a hypersensitivity response in infiltrated tobacco leaves (2,5,19,34). After vigorous proliferation in intercellular spaces, OE1-1 invaded xylem vessels and then systemically infected the whole plant (17,32,33). The hrp mutants of OE1-1 lose their ability not only to proliferate in intercellular spaces but also to systemically infect the whole plant. Therefore, it is thought that proliferation of the bacteria in intercellular spaces after invasion of roots qualitatively control bacterial virulence.The hrp genes encode proteins that construct the type III secretion system. In R. solanacearum, expression of hrp genes is regulated by the HrpB protein (2, 13, 34). Screening of genes controlled by HrpB has isolated many candidates for type III effector genes in R. solanacearum (9,25). Moreover, by in vivo expression technology, genes expressed in R. solanacearum K60-infected tomato plants were isolated (6). However, the involvement of these candidates in the bacterium-host plant interactions remains to be elucidated. Furthermore, analysis by in vivo expression technology indicates that genes other than hrp and type III secretion system effector genes are involved in bacterial virulence (6, 7).To elucidate the mechanism of vigorous growth of OE1-1 in roots in this study, we first selected mutants that lack systemic infectivity and do not provoke disease in tobacco plants. The suicide vector pUTSm/Sp (10) containing mini-Tn5, which includes the spectinomycin resistance gene, was transferred from Escherichia coli HB101 (Takara, Ohtsu, Japan) to OE1-1 by conjugation with E. coli HB101(pRK2013) (12). The roots of 8-week-old tobacco plants (Nicotiana tabacum cv. Bright Yellow) were soaked in a bacterial suspension (1.0 ϫ 10 8 CFU/ml) of 421 spectinomycin-resistant mutants derived from OE1-1 for 30 min, and then inoculated plants were grown in water culture pots (Yamato water culture pot no. 1; Yamato Plastic Co. Ltd., Tokyo, Japan) with one-fifth-strength Hoagland's solution in a growth room at 25°C under 10,000 lx for 16 h/day (root dipping) (17). Each assay was repeated in five successive trials, and within each trial we treated five plants with each strain. Only one mutant, OE1101, lacked the ability to cause wilt in tobacco plants. Seven days after inoculation by root dipping, the stems of five tobacco plants were cut into three pieces with razor blades (Fig. 1). The cut site of each piece was pressed onto Hara-Ono medium (15) for OE1-...

Light modulates important physiological features of Ralstonia pseudosolanacearum during the colonization of tomato plants

“…Multiple alignments of the deduced amino acid sequences of LOV proteins from representative strains belonging to the four phylotypes including: Rpso GMI1000 (phylotype I) 23 , Rpso strain OE1-1 (phylotype I) 35 , Rpso FQY_4 (phylotype I) 36 , Rso K60 (phylotype IIA) 37 , Rso CFBP2957 (phylotype IIA) 38 , Rso IPO1609 (phylotype IIB) 38 , Rso UW551 (phylotype IIB) 38 , Rso Po82 (phylotype IIB) 39 , Rso UY331 (phylotype IIB) 40 , Rpso CMR15 (phylotype III) 38 , R. syzygii R24 (phylotype IV) 41 and R. syzygii PSI07 (phylotype IV) 38 revealed that the LOV protein is present in all Rso strains sequenced and possess highly conserved domains suggesting that light would play an important role in the Rso free lifestyle and during the plant-interaction (Supplementary material S1 ).…”

Contribution of a lectin, LecM, to the quorum sensing signalling pathway of Ralstonia solanacearum strain OE1‐1

“…1, Yamato Plastic Co. Ltd., Yamatotakada, Japan) with fivefold‐diluted Hoagland’s solution. At 10 days post‐inoculation, after sterilization of plant surfaces with 70% ethanol, the roots and stems of five tomato plants were cut into three pieces each using razor blades (Kanda et al ., ). The cut site of each piece (I, roots; II and III, stems; Fig. )…”

Ralfuranones contribute to mushroom‐type biofilm formation by Ralstonia solanacearum strain OE1‐1

“…The colonies were counted after 2 days of incubation at 30 8C. A plate-printing assay was performed as follows (Kanda et al, 2008). At 10 days post-inoculation by root dipping, the roots and stems of five tomato plants were cut into three pieces each using razor blades.…”

“…Ralstonia solanacearum is a soil-borne bacterium that normally invades plant roots from the soil through wounds or natural openings where secondary roots emerge (Araud-Razou et al, 1998), colonizes intercellular spaces of the root cortex and vascular parenchyma, and eventually enters xylem vessels and spreads up into the stems and leaves through the xylem (Hikichi, 2016;Hikichi et al, 2017;Vasse et al, 1995). This colonization in intercellular spaces is required for the virulence of the wild-type strain of R. solanacearum (OE1-1) (Hikichi, 2016;Hikichi et al, 1999Hikichi et al, , 2017Kanda et al, 2003Kanda et al, , 2008Mori et al, 2016;Shinohara et al, 2005). Following the invasion of the intercellular spaces of tomato plants, strain OE1-1 forms mushroom-shaped biofilms (mushroom-type biofilms, mBFs) on the surfaces of tomato cells adjacent to intercellular spaces, leading to the colonization of intercellular spaces (Hikichi, 2016;Hikichi et al, 2017;Mori et al, 2016).…”