Translocation and Functional Analysis ofPseudomonas savastanoipv.savastanoiNCPPB 3335 Type III Secretion System Effectors Reveals Two Novel Effector Families of thePseudomonas syringaeComplex

Abstract

Pseudomonas savastanoi pv. savastanoi NCPPB 3335 causes olive knot disease and is a model pathogen for exploring bacterial infection of woody hosts. The type III secretion system (T3SS) effector repertoire of this strain includes 31 effector candidates plus two novel candidates identified in this study which have not been reported to translocate into plant cells. In this work, we demonstrate the delivery of seven NCPPB 3335 effectors into Nicotiana tabacum leaves, including three proteins from two novel families of the P. syringae complex effector super-repertoire (HopBK and HopBL), one of which comprises two proteins (HopBL1 and HopBL2) that harbor a SUMO protease domain. When delivered by P. fluorescens heterologously expressing a P. syringae T3SS, all seven effectors were found to suppress the production of defense-associated reactive oxygen species. Moreover, six of these effectors, including the truncated versions of HopAA1 and HopAZ1 encoded by NCPPB 3335, suppressed callose deposition. The expression of HopAZ1 and HopBL1 by functionally effectorless P. syringae pv. tomato DC3000D28E inhibited the hypersensitive response in tobacco and, additionally, expression of HopBL2 by this strain significantly increased its competitiveness in N. benthamiana. DNA sequences encoding HopBL1 and HopBL2 were uniquely detected in a collection of 31 P. savastanoi pv. savastanoi strains and other P. syringae strains isolated from woody hosts, suggesting a relevant role of these two effectors in bacterial interactions with olive and other woody plants.

Suppression of Plant Immune Responses by the Pseudomonas savastanoi pv. savastanoi NCPPB 3335 Type III Effector Tyrosine Phosphatases HopAO1 and HopAO2

“…The suppression of early plant defense responses such as ROS production and callose deposition by HopAO1 and HopAO2 was tested using the heterologous expression system P. fluorescens 55 (Pf55) [pLN18] (Jamir et al, 2004; López-Solanilla et al, 2004; Oh et al, 2010; Matas et al, 2014). This system has been previously used to demonstrate the suppression activity of PTI by other P. savastanoi NCPPB 3335 T3SS effectors (Matas et al, 2014).…”

“…savastanoi NCPPB 3335 was performed by marker exchange mutagenesis as previously described (Matas et al, 2014), and the correct exchange of the hopAO1 gene by the Km resistance cassette was determined by Southern blot analysis.…”

“…The translocation assays were performed as described by Matas et al (2014). This method is based on the construction of fusion proteins between identified T3E and the calmodulin-dependent Cya reporter domain.…”

“…However, knowledge about the infection of woody plants by strains belonging to the genus Pseudomonas lags far behind. Although common features with their herbaceous relatives might be found, it should be taken into account that the T3E repertoire of bacterial pathogens isolated from woody hosts, and their functions, might be conditioned by the specific characteristics of woody hosts (Rodriguez-Palenzuela et al, 2010; Matas et al, 2014; Castaneda-Ojeda et al, 2016; Nowell et al, 2016). …”

“…savastanoi NCPPB 3335, a model pathogen for exploring the bacterial infection of woody hosts, includes 33 T3E, nine of which have been shown to translocate into the plant cell (Rodriguez-Palenzuela et al, 2010; Bardaji et al, 2011; Ramos et al, 2012; Matas et al, 2014; Castaneda-Ojeda et al, 2016). The latest contributions to the study of the function of these effectors have revealed new data concerning the specific role of some of them in interfering with plant defense responses (Matas et al, 2014; Castaneda-Ojeda et al, 2016). Among these T3E candidates, only three are homologs of P. syringae pv.…”