The bacterium Pantoea stewartii uses two different type III secretion systems to colonize its plant host and insect vector

Abstract

Plant- and animal-pathogenic bacteria utilize phylogenetically distinct type III secretion systems (T3SS) that produce needle-like injectisomes or pili for the delivery of effector proteins into host cells. Pantoea stewartii subsp. stewartii (herein referred to as P. stewartii), the causative agent of Stewart's bacterial wilt and leaf blight of maize, carries phylogenetically distinct T3SSs. In addition to an Hrc-Hrp T3SS, known to be essential for maize pathogenesis, P. stewartii has a second T3SS (Pantoea secretion island 2 [PSI-2]) that is required for persistence in its flea beetle vector, Chaetocnema pulicaria (Melsh). PSI-2 belongs to the Inv-Mxi-Spa T3SS family, typically found in animal pathogens. Mutagenesis of the PSI-2 psaN gene, which encodes an ATPase essential for secretion of T3SS effectors by the injectisome, greatly reduces both the persistence of P. stewartii in flea beetle guts and the beetle's ability to transmit P. stewartii to maize. Ectopic expression of the psaN gene complements these phenotypes. In addition, the PSI-2 psaN gene is not required for P. stewartii pathogenesis of maize and is transcriptionally upregulated in insects compared to maize tissues. Thus, the Hrp and PSI-2 T3SSs play different roles in the life cycle of P. stewartii as it alternates between its insect vector and plant host.

Fig 1

Organization of the Pantoea stewartii type III secretion system (T3SS), PSI-2. PSI-2 is aligned with T3SS pathogenicity islands from S. glossinidius (11, 12) and Y. enterocolitica (20, 21). PSI-2 genes that are downregulated by HrpL (Table 3) are indicated by asterisks. Putative gene functions include recombination and transposition (IS630 and rve), transcriptional regulators (cadC, mxiE, and ysaE), secretion apparatus structural (dark gray) and regulatory (light gray) functions (psa, ysa, and ye3543A), secreted effector chaperones (pchA and sycA), and secreted effectors (psp and ysp).

Fig 2

Phylogenetic analysis of type three secretion system (T3SS) ATPases from animal- and plant-associated microbes. Accession numbers for the gene and species names shown are given in Materials and Methods. Numbers to the left of the branches are bootstrap values for 1,000 replications, as outlined in Materials and Methods. Plant-associated microbes are designated by a solid line. The positions of the Pantoea stewartii hrcN from the hrc-hrp T3SS and psaN from PSI-2 are indicated by arrows.

Fig 3

The PSI-2 psaN is required for P. stewartii persistence in the flea beetle gut. (A) Diagram of flea beetle foregut (fg), midgut (mg), hindgut (hg), and Malpighian tubules (mt). (B to G) Immunochemical localization of P. stewartii (green fluorescence) in propidium iodide-stained guts (red fluorescence). Beetles fed on healthy control plants for 2 h (B) and 12 days (C), beetles fed on wild-type (DC283) P. stewartii-infected plants for 2 h (day 0) (D) and 12 h (E), and beetles fed on psaN-deficient (DM5121) P. stewartii for 2 h (day 0) (F) and 12 h (G) are shown. Bars, 100 µm.

Fig 4

Persistence of wild-type and psaN-deficient P. stewartii in the flea beetle gut. Beetles were allowed to feed on maize infected with wild-type (DC283; dark gray) or psaN-deficient (DM5121; light gray) strains of P. stewartii for 2 days and then moved to healthy maize, as outlined in Materials and Methods. (A) Percentage of P. stewartii-infected beetles; (B) relative abundance of bacterial foci in beetle guts (rated on a scale of 0 to 4). Data are mean ± standard errors (SE) for 7 to 9 individuals from three biological replicates. *, significantly different (P < 0.05).

Fig 5

Mutation of psaN reduces persistence of colonization of P. stewartii in flea beetles. Beetles were allowed to feed on maize infected with two wild-type strains of P. stewartii (DC283 and DC440) or a psaN (DM5121) or sapD (DM5107) mutant strain. (B) Genetic complementation of the P. stewartii nonpolar psaN∷aphA3 mutant (DM7003) with a plasmid carrying wild-type PsaN (pDM3007) increases P. stewartii persistence in flea beetles. The number of viable P. stewartii cells was estimated as CFU from two or three beetle samples in each of three independent experiments. Data are mean log CFU per insect ± SE. Letters (a, b, and c) indicate significant differences in the number of CFU on day 10 (A) or 8 (B) (P < 0.05).

Fig 6

The PSI-2 psaN is required for persistent transmission of P. stewartii by flea beetles. Beetles were allowed to feed on maize plants infected with wild-type (DC283), psaN-defective strains (DM5121 and DM7003), or a rescued psaN2∷aphA3/psaN⁺ strain [(DM7003(pDM3007)] for 2 days. Individual maize seedlings were then exposed to five beetles each from day 0 or day 8 after acquisition of bacteria for a 2-day transmission period. Data are means ± SE for five seedlings per treatment in three independent experiments. Asterisks indicate means significantly lower than unmarked bars (P < 0.05).