Potent Attractant for Root-Knot Nematodes in Exudates from Seedling Root Tips of Two Host Species.

Root-knot nematodes (RKN; Meloidogyne spp.) can parasitize over 2,000 plant species and are generally considered to be the most agriculturally damaging group of plant-parasitic nematodes worldwide. Infective juveniles (J2) are non-feeding and must locate and invade a host before their reserves are depleted. However, what attracts J2 to appropriate root entry sites is not known. An aim of this research is to identify semiochemicals that attract RKN to roots. J2 of the three RKN species tested are highly attracted to root tips of both tomato and Medicago truncatula. For both hosts, mutants defective in ethylene signaling were found to be more attractive than those of wild type. We determined that cell-free exudates collected from tomato and M. truncatula seedling root tips were highly attractive to M. javanica J2. Using a pluronic gel-based microassay to monitor chemical fractionation, we determined that for both plant species the active component fractionated similarly and had a mass of ~400 based on size-exclusion chromatography. This characterization is a first step toward identification of a potent and specific attractant from host roots that attracts RKN. Such a compound is potentially a valuable tool for developing novel and safe control strategies.

The Type II Secreted Lipase/Esterase LesA is a Key Virulence Factor Required for Xylella fastidiosa Pathogenesis in Grapevines.

Pierce's disease (PD) of grapevines is caused by Xylella fastidiosa (Xf), a xylem-limited gamma-proteobacterium that is responsible for several economically important crop diseases. The occlusion of xylem elements and interference with water transport by Xf and its associated biofilm have been posited as the main cause of PD symptom development; however, Xf virulence mechanisms have not been described. Analysis of the Xf secretome revealed a putative lipase/esterase (LesA) that was abundantly secreted in bacterial culture supernatant and was characterized as a protein ortholog of the cell wall-degrading enzyme LipA of Xanthomonas strains. LesA was secreted by Xf and associated with a biofilm filamentous network. Additional proteomic analysis revealed its abundant presence in outer membrane vesicles (OMVs). Accumulation of LesA in leaf regions associated positively with PD symptoms and inversely with bacterial titer. The lipase/esterase also elicited a hypersensitive response in grapevine. Xf lesA mutants were significantly deficient for virulence when mechanically inoculated into grapevines. We propose that Xf pathogenesis is caused by LesA secretion mediated by OMV cargos and that its release and accumulation in leaf margins leads to early stages of observed PD symptoms.

An engineered innate immune defense protects grapevines from Pierce disease.

We postulated that a synergistic combination of two innate immune functions, pathogen surface recognition and lysis, in a protein chimera would lead to a robust class of engineered antimicrobial therapeutics for protection against pathogens. In support of our hypothesis, we have engineered such a chimera to protect against the gram-negative Xylella fastidiosa (Xf), which causes diseases in multiple plants of economic importance. Here we report the design and delivery of this chimera to target the Xf subspecies fastidiosa (Xff), which causes Pierce disease in grapevines and poses a great threat to the wine-growing regions of California. One domain of this chimera is an elastase that recognizes and cleaves MopB, a conserved outer membrane protein of Xff. The second domain is a lytic peptide, cecropin B, which targets conserved lipid moieties and creates pores in the Xff outer membrane. A flexible linker joins the recognition and lysis domains, thereby ensuring correct folding of the individual domains and synergistic combination of their functions. The chimera transgene is fused with an amino-terminal signal sequence to facilitate delivery of the chimera to the plant xylem, the site of Xff colonization. We demonstrate that the protein chimera expressed in the xylem is able to directly target Xff, suppress its growth, and significantly decrease the leaf scorching and xylem clogging commonly associated with Pierce disease in grapevines. We believe that similar strategies involving protein chimeras can be developed to protect against many diseases caused by human and plant pathogens.

Not as they seem.

I recount the early influences that directed me toward a career in research and then describe some efforts investigating Cowpea mosaic virus and the satellite RNA of Tobacco ringspot virus. These descriptions have a common theme of surprise, how things often can be not as they are expected to be. Finally, I examine the widely held belief that a plant transgene derived from a distant taxonomic source presents a greater risk than a transgene derived from a closely related plant and contend that this also is a situation in which things may not be as they initially seem.

Participation of the Cowpea mosaic virus protease in eliciting extreme resistance.

Extreme resistance of Arlington line cowpea (Vigna unguiculata) to Cowpea mosaic virus (CPMV) is under control of a dominant locus designated Cpa. We transiently expressed, using Tomato bushy stunt virus (TBSV) vectors and Agrobacterium tumefaciens, in nearly isogenic Cpa/Cpa and cpa/cpa cowpea lines, sequences from RNA1, the larger of two CPMV genomic RNAs. Activation of a Cpa-specific response mapped to the CPMV 24K protease (24KPro). Mutational analysis of the 24KPro gene implicated protease activity, rather than 24KPro structure, in Cpa-mediated recognition of CPMV invasion. A 24KPro with alanine replacing the active site cysteine [24KPro(C-A)], but not wildtype 24KPro, accumulated after agroinfiltration of the corresponding binary vector constructions into Cpa/Cpa cowpea. In cpa/cpa cowpea, both protease versions accumulated, with 24KPro(C-A) in greater abundance. Thus, enzymically active 24KPro was recognized by both cowpea genotypes, but in Cpa/Cpa cowpea the suppression of 24KPro accumulation was very strong, consistent with extreme resistance to CPMV.

The Pinus taeda genome is characterized by diverse and highly diverged repetitive sequences.

BACKGROUND: In today's age of genomic discovery, no attempt has been made to comprehensively sequence a gymnosperm genome. The largest genus in the coniferous family Pinaceae is Pinus, whose 110-120 species have extremely large genomes (c. 20-40 Gb, 2N = 24). The size and complexity of these genomes have prompted much speculation as to the feasibility of completing a conifer genome sequence. Conifer genomes are reputed to be highly repetitive, but there is little information available on the nature and identity of repetitive units in gymnosperms. The pines have extensive genetic resources, with approximately 329000 ESTs from eleven species and genetic maps in eight species, including a dense genetic map of the twelve linkage groups in Pinus taeda. RESULTS: We present here the Sanger sequence and annotation of ten P. taeda BAC clones and Genome Analyzer II whole genome shotgun (WGS) sequences representing 7.5% of the genome. Computational annotation of ten BACs predicts three putative protein-coding genes and at least fifteen likely pseudogenes in nearly one megabase of sequence. We found three conifer-specific LTR retroelements in the BACs, and tentatively identified at least 15 others based on evidence from the distantly related angiosperms. Alignment of WGS sequences to the BACs indicates that 80% of BAC sequences have similar copies (> or = 75% nucleotide identity) elsewhere in the genome, but only 23% have identical copies (99% identity). The three most common repetitive elements in the genome were identified and, when combined, represent less than 5% of the genome. CONCLUSIONS: This study indicates that the majority of repeats in the P. taeda genome are 'novel' and will therefore require additional BAC or genomic sequencing for accurate characterization. The pine genome contains a very large number of diverged and probably defunct repetitive elements. This study also provides new evidence that sequencing a pine genome using a WGS approach is a feasible goal.

Determination of preferred pH for root-knot nematode aggregation using pluronic F-127 gel.

Root-knot nematodes (Meloidogyne spp.) are obligate endoparasites of a wide range of plant species. The infective stage is attracted strongly to and enters host roots at the zone of elongation, but the compounds responsible for this attraction have not been identified. We developed a simple assay to investigate nematode response to chemical gradients that uses Pluronic F-127, a synthetic block copolymer that, as a 23% aqueous solution, forms a liquid at low temperature and a gel at room temperature. Test chemicals are put into a modified pipette tip, or 'chemical dispenser,' and dispensers are inserted into the gel in which nematodes have been dispersed. Meloidogyne hapla is attracted to pH gradients formed by acetic acid and several other Brønsted acids and aggregates between pH 4.5 and 5.4. While this pH range was attractive to all tested root-knot nematode strains and species, the level of aggregation depended on the species/strain assessed. For actively growing roots, the pH at the root surface is most acidic at the zone of elongation. This observation is consistent with the idea that low pH is an attractant for nematodes. Root-knot nematodes have been reported to be attracted to carbon dioxide, but our experiments suggest that the observed attraction may be due to acidification of solutions by dissolved CO(2) rather than to CO(2) itself. These results suggest that Pluronic F-127 gel will be broadly applicable for examining responses of a range of organisms to chemical gradients or to each other.

Rapid clearance of bacteria and their toxins: development of therapeutic proteins.

The emergence of pathogens and toxins with resistance against conventional drugs, vaccines, and host defense peptides and proteins warrants novel countermeasures that can efficiently capture and rapidly clear them. This article examines the utility of chimeric proteins with capture and clearance domains as a novel countermeasure against pathogens and their toxins. The capture and clearance domains are chosen from the large repertoire of host defense peptides and proteins. Although individual capture and clearance domains are rendered ineffective by pathogenic resistance mechanisms, chimeric scaffolds can be designed to retain their antimicrobial activity, even in the face of pathogenic resistance. Here, initial studies on the design of chimeric proteins targeted against (1) intact bacteria such as Xylella fastidiosa (plant pathogens), Salmonella spp. (food-borne pathogens), and Staphylococcus aureus (blood-borne pathogens); and (2) lethal toxins from Bacillus anthracis are described.