PhiC31 recombination system demonstrates heritable germinal transmission of site-specific excision from the Arabidopsis genome

<p>Abstract</p> <p>Background</p> <p>The large serine recombinase phiC31 from broad host range <it>Streptomyces </it>temperate phage, catalyzes the site-specific recombination of two recognition sites that differ in sequence, typically known as attachment sites <it>attB </it>and <it>attP</it>. Previously, we characterized the phiC31 catalytic activity and modes of action in the fission yeast <it>Schizosaccharomyces pombe</it>.</p> <p>Results</p> <p>In this work, the <it>phiC31 </it>recombinase gene was placed under the control of the <it>Arabidopsis OXS3 </it>promoter and introduced into <it>Arabidopsis </it>harboring a chromosomally integrated <it>attB </it>and <it>attP</it>-flanked target sequence. The phiC31 recombinase excised the <it>attB </it>and <it>attP</it>-flanked DNA, and the excision event was detected in subsequent generations in the absence of the <it>phiC31 </it>gene, indicating germinal transmission was possible. We further verified that the genomic excision was conservative and that introduction of a functional recombinase can be achieved through secondary transformation as well as manual crossing.</p> <p>Conclusion</p> <p>The phiC31 system performs site-specific recombination in germinal tissue, a prerequisite for generating stable lines with unwanted DNA removed. The precise site-specific deletion by phiC31 <it>in planta </it>demonstrates that the recombinase can be used to remove selectable markers or other introduced transgenes that are no longer desired and therefore can be a useful tool for genome engineering in plants.</p

Localization of DIR1 at the tissue, cellular and subcellular levels during Systemic Acquired Resistance in Arabidopsis using DIR1:GUS and DIR1:EGFP reporters

<p>Abstract</p> <p>Background</p> <p>Systemic Acquired Resistance (SAR) is an induced resistance response to pathogens, characterized by the translocation of a long-distance signal from induced leaves to distant tissues to prime them for increased resistance to future infection. DEFECTIVE in INDUCED RESISTANCE 1 (DIR1) has been hypothesized to chaperone a small signaling molecule to distant tissues during SAR in <it>Arabidopsis</it>.</p> <p>Results</p> <p>DIR1 promoter:DIR1-GUS/<it>dir1-1 </it>lines were constructed to examine DIR1 expression. DIR1 is expressed in seedlings, flowers and ubiquitously in untreated or mock-inoculated mature leaf cells, including phloem sieve elements and companion cells. Inoculation of leaves with SAR-inducing avirulent or virulent <it>Pseudomonas syringae </it>pv <it>tomato </it>(<it>Pst</it>) resulted in Type III Secretion System-dependent suppression of DIR1 expression in leaf cells. Transient expression of fluorescent fusion proteins in tobacco and intercellular washing fluid experiments indicated that DIR1's ER signal sequence targets it for secretion to the cell wall. However, DIR1 expressed without a signal sequence rescued the <it>dir1-1 </it>SAR defect, suggesting that a cytosolic pool of DIR1 is important for the SAR response.</p> <p>Conclusions</p> <p>Although expression of DIR1 decreases during SAR induction, the protein localizes to all living cell types of the vasculature, including companion cells and sieve elements, and therefore DIR1 is well situated to participate in long-distance signaling during SAR.</p

Novel sulI binary vectors enable an inexpensive foliar selection method in Arabidopsis

<p>Abstract</p> <p>Background</p> <p>Sulfonamide resistance is conferred by the <it>sul</it>I gene found on many <it>Enterobacteriaceae </it>R plasmids and Tn21 type transposons. The <it>sul</it>I gene encodes a sulfonamide insensitive dihydropteroate synthase enzyme required for folate biosynthesis. Transformation of tobacco, potato or <it>Arabidopsis </it>using <it>sul</it>I as a selectable marker generates sulfadiazine-resistant plants. Typically <it>sul</it>I-based selection of transgenic plants is performed on tissue culture media under sterile conditions.</p> <p>Findings</p> <p>A set of novel binary vectors containing a <it>sul</it>I selectable marker expression cassette were constructed and used to generate transgenic <it>Arabidopsis</it>. We demonstrate that the <it>sul</it>I selectable marker can be utilized for direct selection of plants grown in soil with a simple foliar spray application procedure. A highly effective and inexpensive high throughput screening strategy to identify transgenic <it>Arabidopsis </it>without use of tissue culture was developed.</p> <p>Conclusion</p> <p>Novel <it>sul</it>I-containing <it>Agrobacterium </it>binary vectors designed to over-express a gene of interest or to characterize a test promoter in transgenic plants have been constructed. These new vector tools combined with the various beneficial attributes of sulfonamide selection and the simple foliar screening strategy provide an advantageous alternative for plant biotechnology researchers. The set of binary vectors is freely available upon request.</p

Quantitative and Qualitative Stem Rust Resistance Factors in Barley Are Associated with Transcriptional Suppression of Defense Regulons

Stem rust (Puccinia graminis f. sp. tritici; Pgt) is a devastating fungal disease of wheat and barley. Pgt race TTKSK (isolate Ug99) is a serious threat to these Triticeae grain crops because resistance is rare. In barley, the complex Rpg-TTKSK locus on chromosome 5H is presently the only known source of qualitative resistance to this aggressive Pgt race. Segregation for resistance observed on seedlings of the Q21861 × SM89010 (QSM) doubled-haploid (DH) population was found to be predominantly qualitative, with little of the remaining variance explained by loci other than Rpg-TTKSK. In contrast, analysis of adult QSM DH plants infected by field inoculum of Pgt race TTKSK in Njoro, Kenya, revealed several additional quantitative trait loci that contribute to resistance. To molecularly characterize these loci, Barley1 GeneChips were used to measure the expression of 22,792 genes in the QSM population after inoculation with Pgt race TTKSK or mock-inoculation. Comparison of expression Quantitative Trait Loci (eQTL) between treatments revealed an inoculation-dependent expression polymorphism implicating Actin depolymerizing factor3 (within the Rpg-TTKSK locus) as a candidate susceptibility gene. In parallel, we identified a chromosome 2H trans-eQTL hotspot that co-segregates with an enhancer of Rpg-TTKSK-mediated, adult plant resistance discovered through the Njoro field trials. Our genome-wide eQTL studies demonstrate that transcript accumulation of 25% of barley genes is altered following challenge by Pgt race TTKSK, but that few of these genes are regulated by the qualitative Rpg-TTKSK on chromosome 5H. It is instead the chromosome 2H trans-eQTL hotspot that orchestrates the largest inoculation-specific responses, where enhanced resistance is associated with transcriptional suppression of hundreds of genes scattered throughout the genome. Hence, the present study associates the early suppression of genes expressed in this host–pathogen interaction with enhancement of R-gene mediated resistance

The function of the tomato Pto gene in tobacco and the identification and characterization of two genes induced by the Pto pathway

The Pto gene encodes a serine-threonine kinase that confers resistance in tomato to Pseudomonas syringae pv. tomato strains expressing the avirulence gene avrPto. I examined the ability of Pto to function in tobacco. The Pto-transformed tobacco plants exhibited a significant increase in resistance to the avirulent P. s. tabaci strain compared with wild-type tobacco as indicated by: (1) more rapid development of the hypersensitive response (HR) at high inoculum concentrations; (2) lessened severity of disease symptoms at moderate inoculum concentrations; and (3) reduced growth of avindent P. s. tabaci in inoculated leaves. The results indicate that essential components of a Pto-mediated signal transduction pathway are conserved in tobacco. Salicylic acid (SA) is an important signal molecule involved in the plant disease resistance. I examined the role of SA in Pto mediated resistance using tobacco plants that express both the Pto and the nahG transgenes. The nahG gene, which encodes salicylate hydroxylase, attenuates local resistance and blocks systemic acquired resistance in tobacco and Arabidopsis. Inoculated tobacco leaves expressing Pto and nahG exhibit an increase in symptom development and avirulent bacterial growth compared to tobacco plants expressing Pto alone. This reduction in resistance is correlated with a delay in the expression of pathogenesis related (PR) genes within inoculated tissue. Despite these changes in resistance, the Pto/nahG plants exhibit an unaltered HR and allow less bacterial growth in their leaves than nahG plants that lacked the Pto transgene. In addition, Pto/ nahG plants express the hin1 and hsr203J genes to similar levels as control plants. Expression of nahG effectively blocked systemic acquired resistance and systemic PR gene expression that is activated by the Pto-mediated HR. These results suggest that Pto-mediated resistance involves both SA dependent and independent pathways. The Pto-mediated defense response involves the induction of PR genes and potentially other defense genes. I used the mRNA differential display technique to identify two genes in tomato that are specifically induced by the Pto pathway. Transcripts of Prg1 (Pto responsive gene 1) and Le-Eli3 rapidly accumulate in Pto-resistant plants inoculated with the avirulent pathogen. Prg1 is a single copy gene and lacks homology to any known proteins in the database. Le-Eli3 is a member of a gene family in tomato and has significant homology to the ELI3 defense response genes from Arabidopsis and parsley. The expression of Prg1 and Le-Eli3 in response to SA and their developmental expression in different tomato tissues was examined

Escherichia coli O157:H7 Converts Plant-Derived Choline to Glycine Betaine for Osmoprotection during Pre- and Post-harvest Colonization of Injured Lettuce Leaves

Plant injury is inherent to the production and processing of fruit and vegetables. The opportunistic colonization of damaged plant tissue by human enteric pathogens may contribute to the occurrence of outbreaks of foodborne illness linked to produce. Escherichia coli O157:H7 (EcO157) responds to physicochemical stresses in cut lettuce and lettuce lysates by upregulation of several stress response pathways. We investigated the tolerance of EcO157 to osmotic stress imposed by the leakage of osmolytes from injured lettuce leaf tissue. LC-MS analysis of bacterial osmoprotectants in lettuce leaf lysates and wound washes indicated an abundant natural pool of choline, but sparse quantities of glycine betaine and proline. Glycine betaine was a more effective osmoprotectant than choline in EcO157 under osmotic stress conditions in vitro. An EcO157 mutant with a deletion of the betTIBA genes, which are required for biosynthesis of glycine betaine from imported choline, achieved population sizes twofold lower than those of the parental strain (P &lt; 0.05) over the first hour of colonization of cut lettuce in modified atmosphere packaging (MAP). The cell concentrations of the betTIBA mutant also were 12-fold lower than those of the parental strain (P &lt; 0.01) when grown in hypertonic lettuce lysate, indicating that lettuce leaf cellular contents provide choline for osmoprotection of EcO157. To demonstrate the utilization of available choline by EcO157 for osmoadaptation in injured leaf tissue, deuterated (D-9) choline was introduced to wound sites in MAP lettuce; LC-MS analysis revealed the conversion of D9-choline to D-9 glycine betaine in the parental strain, but no significant amounts were observed in the betTIBA mutant. The EcO157 ΔbetTIBA-ΔotsBA double mutant, which is additionally deficient in de novo synthesis of the compatible solute trehalose, was significantly less fit than the parental strain after their co-inoculation onto injured lettuce leaves and MAP cut lettuce. However, its competitive fitness followed a different time-dependent trend in MAP lettuce, likely due to differences in O2 content, which modulates betTIBA expression. Our study demonstrates that damaged lettuce leaf tissue does not merely supply EcO157 with substrates for proliferation, but also provides the pathogen with choline for its survival to osmotic stress experienced at the site of injury

CRISPR-Cas9 Gene Editing of the Sal1 Gene Family in Wheat

The highly conserved Sal1 encodes a bifunctional enzyme with inositol polyphosphate-1-phosphatase and 3&prime; (2&prime;), 5&prime;-bisphosphate nucleotidase activity and has been shown to alter abiotic stress tolerance in plants when disrupted. Precise gene editing techniques were used to generate Sal1 mutants in hexaploid bread wheat. The CRISPR (Clustered Regulatory Interspaced Short Palindromic Repeats) Cas9 system with three guide RNAs (gRNAs) was used to inactivate six Sal1 homologous genes within the Bobwhite wheat genome. The resulting mutant wheat plants with all their Sal1 genes disabled had slimmer stems, had a modest reduction in biomass and senesced more slowly in water limiting conditions, but did not exhibit improved yield under drought conditions. Our results show that multiplexed gRNAs enabled effective targeted gene editing of the Sal1 gene family in hexaploid wheat. These Sal1 mutant wheat plants will be a resource for further research studying the function of this gene family in wheat