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Many strains of Pseudomonas syringae produce retractile pili that act as receptors for lytic bacteriophage phi 6. As these are also characteristics of type IV pili, it was postulated that P. syringae may possess genes for type IV pilus biogenesis. A cosmid clone bank of P. syringae pv. tomato DC3000 genomic DNA was used to complement a mutant of Pseudomonas aeruginosa defective in the PilD (XcpA) prepilin peptidase gene by selection for restoration of extracellular protein secretion, a function also known to require PilD. A cosmid able to complement this mutant was also able to complement mutations in the pilB and pilC genes, suggesting that, if the organization of these genes is similar to that of P. aeruginosa, the cosmid may contain the P. syringae pilA. This was confirmed by sequencing a region from this plasmid that was shown to hybridize at low stringency to the P. aeruginosa pilA gene. The deduced P. syringae PilA polypeptide possesses the characteristic properties of the type IV pilins. Heterologous expression of the P. syringae pilA in P. aeruginosa was also shown, conferring not only phi 6 phage sensitivity to P. aeruginosa pilA mutants but also sensitivity to PO4, a lytic bacteriophage specific for the pilus of P. aeruginosa. This suggests that additional components might be present in the mature pilus of P. aeruginosa that are the true receptors for this phage. Chromosomal mutations in P. syringae pv. tomato DC3000 pilA and pilD genes were shown to abolish its sensitivity to bacteriophage phi 6. To determine the importance of P. syringae pilus in plant leaf interactions, these mutations were tested under laboratory and field conditions. Although little effect was seen on pathogenicity, culturable leaf-associated population sizes of the pilA mutant were significantly different from those of the wild-type parent. In addition, the expression of the DC3000 pilA gene appears to contribute to the UV tolerance of P. syringae and may play a role in survival on the plant leaf surface.

The transferred DNA (T-DNA) is transported from Agrobacterium tumefaciens to the nucleus and is stably integrated into the genome of many plant species. It has been proposed that the VirD2 protein, tightly attached to the T-DNA, pilots the T-DNA into the plant cell nucleus and that it is involved in integration. Using agroinfection and beta-glucuronidase expression as two different very sensitive transient assays for T-DNA transfer, together with assays for stable integration, we have shown that the C-terminal half of the VirD2 protein and the VirD3 protein are not involved in T-DNA integration. However, the bipartite nuclear localization signal, which is located within the C terminus of the VirD2 protein and which has previously been shown to be able to target a foreign protein into the plant cell nucleus, was shown to be required for efficient T-DNA transfer. virD4 mutants were shown by agroinfection to be completely inactive in T-DNA transfer.

Agrobacteria exhibit marked Ti (tumorinducing)/Ri (root-inducing) plasmid specificity in their interaction with the Gramineae. In this study, we have used the technique of "agroinfection," in which Agrobacterium-mediated delivery of viral genomes into plants is detected by the development of viral disease symptoms, to identify the region of the Ti plasmid which is responsible for the major differences seen in the ability of nopaline-vs. plants (4, 5). However, more recent work reported that certain monocotyledonous plants (monocots) are also susceptible (6-9). Furthermore, although maize and other graminaceous monocots have remained refractory to tumor formation, the agroinfection technique used to study the transfer of viral DNA to dicots (10, 11) has confirmed that Agrobacterium is able to transfer DNA to cereal plants (12)(13)(14)(15)(16)(17). Agroinfection is mediated through the same vir gene pathway as is required for tumor formation in dicots (18).This technique provides a very sensitive assay for Agrobacterium-mediated DNA transfer, as the viral sequences escape from the T-DNA and are amplified as the virus replicates and spreads systemically. DNA transfer is thus detected by the formation of disease symptoms.The ability to agroinfect maize was found to be Ti plasmidspecific (13, 16): octopine-type strains either were unable, or only weakly able (<5%), to transfer maize streak virus (MSV) DNA, while nopaline-type strains infected 80-100%o of the inoculated plants. Agropine-and mannopine-type strains of A. rhizogenes also efficiently agroinfected maize. With appropriate viral DNAs as markers, similar results were obtained for other members of the Gramineae (14,15,17). In contrast, Ti plasmid specificity is not typical of agroinfection-dicot interactions (10, 11).A "disarmed" (T-DNA-less) nopaline strain agroinfected maize as efficiently as the wild type (13). Therefore, Ti plasmid specificity cannot be due to differences in phytohormone production specified by the T-DNA. One explanation for the inability of octopine-type strains to agroinfect maize and other graminaceous monocots is that octopine-type Ti plasmids lack an essential function present in nopaline-, agropine-, and mannopine-type Ti/Ri plasmids. Alternatively, octopine-type Ti plasmids might express a counteractive function.We have used the agroinfection system to characterize the Ti plasmid requirements for transfer of MSV DNA to maize. We observed that a DNA fragment containing the nopalinetype virA locus complemented octopine-type Ti plasmids to high levels of agroinfection. Furthermore, whereas preinduction of octopine-type strains does not promote maize agroinfection, octopine-type virA mutant strains, which express vir genes at high levels in the absence of the inducing compound acetosyringone, efficiently transferred MSV DNA to maize. We discuss here the implications of these observations and possible causes. MATERIALS AND METHODSBacterial Strains, Plasmids, and Media. The MSV-containing binary vectors and other plasmids used in...

The ability of Agrobacterium strains to infect transformation-recalcitrant maize plants has been shown to be determined mainly by the virA locus, implicating vir gene induction as the major factor influencing maize infection. In this report, we further explore the roles of vir induction-associated bacterial factors in maize infection using the technique of agroinfection. The Ti plasmid and virA source are shown to be important in determining the ability of a strain to infect maize, and the monosaccharide binding protein ChvE is absolutely required for maize agroinfection. The linker domain of VirAC58 from an agroinfection-competent strain, C58, is sufficient to convert VirAA6 of a nonagroinfecting strain, A348,to agroinfection competence. The periplasmic domain of VirAC58 is also able to confer a moderate level of agroinfection competence to VirAA6. In addition, the VirAA6 protein from A348 is agroinfection competent when removed from its cognate Ti plasmid background and placed in a pTiC58 background. The presence of a pTiA6-encoded, VirAA6-specific inhibitor is hypothesized and examined.