A Pseudomonas syringae pv. tomato DC3000 mutant lacking the type III effector HopQ1‐1 is able to cause disease in the model plant Nicotiana benthamiana

Abstract

The model pathogen Pseudomonas syringae pv. tomato DC3000 causes bacterial speck in tomato and Arabidopsis, but Nicotiana benthamiana, an important model plant, is considered to be a non-host. Strain DC3000 injects approximately 28 effector proteins into plant cells via the type III secretion system (T3SS). These proteins were individually delivered into N. benthamiana leaf cells via T3SS-proficient Pseudomonas fluorescens, and eight, including HopQ1-1, showed some capacity to cause cell death in this test. Four gene clusters encoding 13 effectors were deleted from DC3000: cluster II (hopH1, hopC1), IV (hopD1, hopQ1-1, hopR1), IX (hopAA1-2, hopV1, hopAO1, hopG1), and native plasmid pDC3000A (hopAM1-2, hopX1, hopO1-1, hopT1-1). DC3000 mutants deleted for cluster IV or just hopQ1-1 acquired the ability to grow to high levels and produce bacterial speck lesions in N. benthamiana. HopQ1-1 showed other hallmarks of an avirulence determinant in N. benthamiana: expression in the tobacco wildfire pathogen P. syringae pv. tabaci 11528 rendered this strain avirulent in N. benthamiana, and elicitation of the hypersensitive response in N. benthamiana by HopQ1-1 was dependent on SGT1. DC3000 polymutants involving other effector gene clusters in a hopQ1-1-deficient background revealed that clusters II and IX contributed to the severity of lesion symptoms in N. benthamiana, as well as in Arabidopsis and tomato. The results support the hypothesis that the host ranges of P. syringae pathovars are limited by the complex interactions of effector repertoires with plant anti-effector surveillance systems, and they demonstrate that N. benthamiana can be a useful model host for DC3000

Genetic disassembly and combinatorial reassembly identify a minimal functional repertoire of type III effectors in Pseudomonas syringae

“…We had previously constructed CUCPB5500, which lacks all 18 of the well-expressed T3E genes occurring in clusters (13). Here, we deleted the remaining 10 well-expressed T3E genes, again using pK18mobsacB (11), to produce polymutant DC3000D28E (CUCPB5585 in Fig. 1).…”

“…How these few T3Es function together to form a minimal repertoire may be complex, because T3Es can have multiple domains and interfering activities. For example, AvrPtoB also possesses an E3 ubiquitin ligase domain that can suppress ETI (28), and HopM1, AvrE, and HopAA1-1 elicit ETI-like cell death in N. benthamiana when individually delivered by the nonpathogen P. fluorescens expressing cloned P. syringae T3SS genes (11). An advantage of the DC3000D28E PRIVAS system is that it provides strong phenotypes and facile tools for dissection of T3Es and their interplay in near native settings.…”

“…Bacterial strains and plasmids are described in SI Materials and Methods, Construction and Usage of the Vectors for Genomic Gene Replacement and Complementation and Table S1. Mutations and restorations of complementing genes to native loci were performed with the suicide-eviction vector pK18mobsacB and then confirmed by PCR (11). Primers for plasmid and mutant construction are given in Table S2.…”

“…The majority of the well-expressed DC3000 T3Es are encoded within six clusters in the DC3000 genome (11). Deletions of individual clusters revealed HopQ1-1 to function as the sole avirulence determinant for DC3000 in N. benthamiana, a plant that is particularly amenable to high-throughput genetic manipulation and bacterial growth assays (11,12).…”

“…Deletions of individual clusters revealed HopQ1-1 to function as the sole avirulence determinant for DC3000 in N. benthamiana, a plant that is particularly amenable to high-throughput genetic manipulation and bacterial growth assays (11,12). Combinatorial deletions revealed only a small reduction in growth in N. benthamiana with the loss of 15 T3E genes in five clusters but a stronger reduction with the loss of just two or three T3E genes in either of two redundant effector groups (REGs).…”