A luminescent reporter evidences active expression of Ralstonia solanacearum type III secretion system genes throughout plant infection

Although much is known about the signals that trigger transcription of virulence genes in plant pathogens, their prevalence and timing during infection are still unknown. In this work, we address these questions by analysing expression of the main pathogenicity determinants in the bacterial pathogen Ralstonia solanacearum. We set up a quantitative, non-invasive luminescent reporter to monitor in planta transcription from single promoters in the bacterial chromosome. We show that the new reporter provides a real-time measure of promoter output in vivo – either after re-isolation of pathogens from infected plants or directly in situ – and confirm that the promoter controlling exopolysaccharide (EPS) synthesis is active in bacteria growing in the xylem. We also provide evidence that hrpB, the master regulator of type III secretion system (T3SS) genes, is transcribed in symptomatic plants. Quantitative RT-PCR assays demonstrate that hrpB and type III effector transcripts are abundant at late stages of plant infection, suggesting that their function is required throughout disease. Our results challenge the widespread view in R. solanacearum pathogenicity that the T3SS, and thus injection of effector proteins, is only active to manipulate plant defences at the first stages of infection, and that its expression is turned down when bacteria reach high cell densities and EPS synthesis starts.

F. M. held a PhD fellowship from the Fundação para a Ciência e a Tecnologia (RFRH/BD/45850/2008). This work was supported by grants from Comissionat per Universitats i Recerca of the Generalitat de Catalunya (SGR0052 and CONES2010-0030) and from the Ministerio de Ciencia, Tecnología e Innovación of the Spanish Government (HF2008-0021 and AGL2010-21870). S. G. is part of the ‘Laboratoire d’Excellence’ (LABEX) entitled TULIP (ANR-10-LABX-41).

Peer reviewed