Pseudomonas syringae pv. tomato DC3000 polymutants deploying coronatine and two type III effectors produce quantifiable chlorotic spots from individual bacterial colonies in Nicotiana benthamiana leaves

Abstract

Primary virulence factors of Pseudomonas syringae pv. tomato DC3000 include the phytotoxin coronatine (COR) and a repertoire of 29 effector proteins injected into plant cells by the type III secretion system (T3SS). DC3000 derivatives differentially producing COR, the T3SS machinery and subsets of key effectors were constructed and assayed in leaves of Nicotiana benthamiana. Bacteria were inoculated by the dipping of whole plants and assayed for population growth and the production of chlorotic spots on leaves. The strains fell into three classes. Class I strains are T3SS but functionally effectorless, grow poorly in planta and produce faint chlorotic spots only if COR . Class II strains are T3SS or, if T3SS , also produce effectors AvrPtoB and HopM1. Class II strains grow better than class I strains in planta and, if COR , produce robust chlorotic spots. Class III strains are T3SS and minimally produce AvrPtoB, HopM1 and three other effectors encoded in the P. syringae conserved effector locus. These strains differ from class II strains in growing better in planta, and produce chlorotic spots without COR if the precursor coronafacic acid is produced. Assays for chlorotic spot formation, in conjunction with pressure infiltration of low-level inoculum and confocal microscopy of fluorescent protein-labelled bacteria, revealed that single bacteria in the apoplast are capable of producing colonies and associated leaf spots in a 1 : 1 : 1 manner. However, COR makes no significant contribution to the bacterial colonization of the apoplast, but, instead, enables a gratuitous, semi-quantitative, surface indicator of bacterial growth, which is determined by the strain's effector composition.

Ca 2+ -Induced Two-Component System CvsSR Regulates the Type III Secretion System and the Extracytoplasmic Function Sigma Factor AlgU in Pseudomonas syringae pv. tomato DC3000

“…Decreased spread of chlorotic symptoms by P. syringae pv. tomato DC3000 commonly occurs as a result of changes in T3Es or coronatine production (71)(72)(73). While we have not investigated whether CvsSR regulates coronatine production in P. syringae pv.…”

Defining essential processes in plant pathogenesis with Pseudomonas syringae pv. tomato DC3000 disarmed polymutants and a subset of key type III effectors

“…). Disassembly occurred with the following major steps: (i) hopQ1‐1 was deleted, which expanded the host range to the model plant N. benthamiana (Wei et al ., ); (ii) 18 T3E genes located in five genomic clusters and plasmid pDC3000A were deleted, as groups in various combinations (Kvitko et al ., ; Wei et al ., ); (iii) the remaining 10 well‐expressed T3E genes that reside outside of these clusters were sequentially deleted to produce DC3000D28E (here abbreviated ‘D28E’) (Cunnac et al ., ); (iv) a reassembly process involving the shuffling of small sets of T3E genes that were assayed for promotion of growth and symptoms in pressure‐infiltrated N. benthamiana revealed a subset of eight effectors sufficient for disease (Cunnac et al ., ); (v) hopAD , which was considered as weakly expressed (Chang et al ., ), was subsequently deleted from D28E to produce ‘D29E’ and thereby to eliminate residual elicitation of cell death in N. benthamiana at very high levels of inoculum (Wei et al ., ); (vi) additional weakly expressed T3E genes were deleted to produce ‘D36E’ (Wei et al ., ); (vii) further analysis of combinations of T3Es in the minimal repertoire, using dip inoculations and a D29E derivative restored for COR production (explained below), revealed that just two T3Es — HopM1 and AvrPtoB — were sufficient to produce robust chlorotic spots in N. benthamiana (Chakravarthy et al ., ). Finally, it is worth noting that there is no apparent difference in the response of N. benthamiana to the effector‐depleted polymutants D29E and D36E (deleted of all known T3E genes and several pseudogenes) (Wei et al ., ).…”

“…In general, DC3000 and derivatives varying in their production of these key T3Es can be divided into four phenotypic classes (Chakravarthy et al ., ). Class I strains do not grow in N. benthamiana and are represented by the effector‐depleted polymutants D28E, D29E and D36E.…”

“… HopM1 (Baltrus et al ., ; Cai et al ., ; Chakravarthy et al ., ; Debroy et al ., ; Lozano‐Duran et al ., , Nomura et al ., ; Ustun et al ., ; Xin et al ., ).…”

“…MAMPs trigger stomatal closure as part of the PTI response, and the virulence in Arabidopsis of surface‐inoculated Pst DC3000 strongly benefits from the phytotoxin COR, a jasmonic acid‐isoleucine (JA‐Ile) analogue that reverses stomatal closure (Melotto et al ., ). COR also promotes symptoms in Pst DC3000 hosts (discussed further below), but appears to be less important for DC3000 entry into N. benthamiana leaves, and is produced by only a subset of P. syringae pathovars (Chakravarthy et al ., ; O'Brien et al ., ). Some Pst DC3000 T3Es, including HopM1, HopF2 and HopX1, also have the potential to block stomatal immunity (Gimenez‐Ibanez et al ., ; Hurley et al ., ; Lozano‐Duran et al ., ).…”