Analysis of the Role of the Type III Effector Inventory ofPseudomonas syringaepv. phaseolicola 1448a in Interaction with the Plant

Abstract

In Pseudomonas syringae, the type III secretion system (T3SS) is essential for disease in compatible hosts and for eliciting the hypersensitive response in incompatible hosts. P. syringae pathovars secrete a variable number of type III effectors that form their secretomes. The secretome of Pseudomonas syringae pv. phaseolicola 1448a (Pph1448a) currently includes 22 experimentally validated effectors, one HrpL-regulated candidate for which translocation results have been inconsistent, two translocated candidates for which in planta expression has not been established, one bioinformatically identified candidate, and six candidates that have been experimentally discarded. We analyzed the translocation and/or expression of these and other candidates to complete the Pph1448a effector inventory, bringing this inventory to 27 bona fide effectors, including a new one that does not belong to any of the previously described effector families. We developed a simple process for rapidly making single and double knockout mutants and apply it to the generation of an effector mutant collection that includes single knockouts for the majority of the Pph1448a effector inventory. We also generated two double mutant strains containing effectors with potentially redundant functions and analyzed the virulence of the single and double mutant strains as well as strains expressing each of the effectors from a plasmid. We demonstrate that AvrB4-1 and AvrB4-2, as well as HopW1-1 and HopW1-2, are fully redundant and contribute to virulence in bean plants, thus validating this approach for dissecting the contribution of the Pph1448a type III effector inventory to virulence. We also analyzed the effect that the expression of these four effectors from Pseudomonas syringae pv. tomato DC3000 (PtoDC3000) has during its interaction with Arabidopsis thaliana, establishing that AvrB4-1, but not the others, determines a restriction of bacterial growth that takes place mostly independently of the salicylic acid (SA)-signaling pathway.

The Pseudomonas aeruginosa PA14 ABC Transporter NppA1A2BCD Is Required for Uptake of Peptidyl Nucleoside Antibiotics

“…Construction of the PA14 nppBCD knockout mutant. The construction of the knockout vector was based on the protocol described by Zumaquero et al (12). Briefly, approximately 500-bp sequences flanking the 5= and 3= regions of the nppBCD operon were PCR amplified with primers 41130-A1 and 41130-A2 and primers 41170-B1 and 41170-B2.…”

AraC/XylS Family Stress Response Regulators Rob, SoxS, PliA, and OpiA in the Fire Blight Pathogen Erwinia amylovora

“…Briefly, a 1,220-bp fragment flanking the 5= region and a 777-bp fragment flanking the 3= region of the opiA gene were PCR amplified using primer pairs opiA-A5-1-opiA-A6 and opiA-B3-opiA-B4, respectively. Primers opiA-A6 and opiA-B3 share a 20-nucleotide homologous sequence at their 5= ends, consisting of the T7 primer sequence and a KpnI restriction site (41). The obtained fragments were gel purified, and approximately 40 ng (each) of A and B fragments was used for a fusion PCR with primers opiA-A5 and opiA-B4.…”

“…The construction of the opiA knockout vector was based on the protocol described by Zumaquero et al (41). Briefly, a 1,220-bp fragment flanking the 5= region and a 777-bp fragment flanking the 3= region of the opiA gene were PCR amplified using primer pairs opiA-A5-1-opiA-A6 and opiA-B3-opiA-B4, respectively.…”

The Molecular Basis of Host Specialization in Bean Pathovars of Pseudomonas syringae

“…phaseolicola 1448a (Fig. 3A) (Baltrus et al 2011;Chang et al 2005;Zumaquero et al 2010). There were 13 T3E genes shared by all three isolates, as well as one conserved truncation within hopAA1.…”