Rapid in vivo analysis of synthetic promoters for plant pathogen phytosensing

<p>Abstract</p> <p>Background</p> <p>We aimed to engineer transgenic plants for the purpose of early detection of plant pathogen infection, which was accomplished by employing synthetic pathogen inducible promoters fused to reporter genes for altered phenotypes in response to the pathogen infection. Toward this end, a number of synthetic promoters consisting of inducible regulatory elements fused to a red fluorescent protein (RFP) reporter were constructed for use in phytosensing.</p> <p>Results</p> <p>For rapid analysis, an <it>Agrobacterium</it>-mediated transient expression assay was evaluated, then utilized to assess the inducibility of each synthetic promoter construct <it>in vivo</it>. Tobacco (<it>Nicotiana tabacum </it>cv. Xanthi) leaves were infiltrated with <it>Agrobacterium </it>harboring the individual synthetic promoter-reporter constructs. The infiltrated tobacco leaves were re-infiltrated with biotic (bacterial pathogens) or abiotic (plant defense signal molecules salicylic acid, ethylene and methyl jasmonate) agents 24 and 48 hours after initial agroinfiltration, followed by RFP measurements at relevant time points after treatment. These analyses indicated that the synthetic promoter constructs were capable of conferring the inducibility of the RFP reporter in response to appropriate phytohormones and bacterial pathogens, accordingly.</p> <p>Conclusions</p> <p>These observations demonstrate that the <it>Agrobacterium</it>-mediated transient expression is an efficient method for <it>in vivo </it>assays of promoter constructs in less than one week. Our results provide the opportunity to gain further insights into the versatility of the expression system as a potential tool for high-throughput <it>in planta </it>expression screening prior to generating stably transgenic plants for pathogen phytosensing. This system could also be utilized for temporary phytosensing; e.g., not requiring stably transgenic plants.</p

Octopine- and nopaline-inducible proteins in Agrobacterium tumefaciens are also induced by arginine

Octopine induced the synthesis of 83, 76, 62, 58, 44, 42, 31, and 22 kDa proteins in Agrobacterium tumefaciens strains harboring the tumor-inducing (Ti) plasmids pTiA6 and pTiAch5. Nopaline induced the synthesis of 83, 76, 62, 58, 56, 44, 42, 31, and 22 kDa proteins in A. tumefaciens strains harboring the Ti plasmids pTiC58 and pTiT37. The molecular masses of proteins induced by octopine and nopaline were very similar. In accordance with the 'opine concept', octopine and nopaline were found to induce protein synthesis only in strains harboring the respective Ti plasmids. Arginine, a common catabolic product of octopine and nopaline, induced the synthesis of most of the proteins induced by the two opines. Our results show that only the initial step(s) of octopine and nopaline catabolism are induced by specific opines in the respective strains. The subsequent steps are likely to be regulated by arginine in both strains

Sorghum Transformation: Overview and Utility

Over the past decade genomics resources available for sorghum have rapidly expanded (Paterson Int J Plant Genomics 2008:6, 2008), these resources, coupled with the recent completion of the genome sequence which is relatively small in size (730 Mb) (Paterson et al. Nature 457:551–556, 2009) makes sorghum a rather attractive species to study. Moreover, the USDA germplasm system maintains 42,614 accessions, of which more than 800 exotic landraces have been converted to day length-insensitive lines to facilitate their use in breeding programs. In addition, a set of EMS mutation stocks developed by the USDA Plant Stress and Germplasm Development Unit in Lubbock, TX (Xin et al. Bioenerg Res 2:10–16, 2009) will be a valuable resource for functional genomics studies in sorghum. However, in order to be a robust system for study a suite of functional genomics tools are necessary to complement these other resources to aid in down-stream hypothesis testing. A key functional genomics tool is the ability to modulate gene expression through the introduction of transgenic genetic elements. This is exemplified by recent work (Cook et al. Plant Cell 22:867–887, 2010) in which RNAi experiments were employed to specifically reduced expression of two alkylresorcinol synthases to demonstrate their role in the synthesis of the allelopathic molecule sorgoleone. In addition to its value as a functional genomics tool, plant transformation offers a route to broaden access to novel input and output traits for sorghum breeding programs

The Agrobacterium tumefaciens rnd Homolog Is Required for TraR-Mediated Quorum-Dependent Activation of Ti Plasmid tra Gene Expression

Conjugal transfer of Agrobacterium tumefaciens Ti plasmids is regulated by quorum sensing via TraR and its cognate autoinducer, N-(3-oxo-octanoyl)-l-homoserine lactone. We isolated four Tn5-induced mutants of A. tumefaciens C58 deficient in TraR-mediated activation of tra genes on pTiC58ΔaccR. These mutations also affected the growth of the bacterium but had no detectable influence on the expression of two tester gene systems that are not regulated by quorum sensing. In all four mutants Tn5 was inserted in a chromosomal open reading frame (ORF) coding for a product showing high similarity to RNase D, coded for by rnd of Escherichia coli, an RNase known to be involved in tRNA processing. The wild-type allele of the rnd homolog cloned from C58 restored the two phenotypes to each mutant. Several ORFs, including a homolog of cya2, surround A. tumefaciens rnd, but none of these genes exerted a detectable effect on the expression of the tra reporter. In the mutant, traR was expressed from the Ti plasmid at a level about twofold lower than that in NT1. The expression of tra, but not the growth rate, was partially restored by increasing the copy number of traR or by disrupting traM, a Ti plasmid gene coding for an antiactivator specific for TraR. The mutation in rnd also slightly reduced expression of two tested vir genes but had no detectable effect on tumor induction by this mutant. Our data suggest that the defect in tra gene induction in the mutants results from lowered levels of TraR. In turn, production of sufficient amounts of TraR apparently is sensitive to a cellular function requiring RNase D