Evaluation of novel surfactants for plant transformation.

OBJECTIVE: Assess the efficiency of seven novel surfactants, relative to the typical Silwet® L-77, for floral dip transformation of Arabidopsis. RESULTS: Floral dip transformation of Arabidopsis has been used consistently for 20 years with little change in the protocol. Here we directly compare seven novel surfactants (BREAK-THRU®-OE446, S200, S233, S240, S279, S301 and SP133) to the standard Silwet® L-77 for efficiency of Arabidopsis transformation providing an example of how the surfactants can help other plant transformation protocols. Relative transformation efficiencies ranged from - 44 to + 45% compared to Silwet® L-77. Surfactants S200, S240, and S279 demonstrated the greatest enhancement in transformation.

Isolation of novel citrus and plum fruit promoters and their functional characterization for fruit biotechnology.

BACKGROUND: Promoters that confer expression in fruit tissues are important tools for genetic engineering of fruit quality traits, yet few fruit-specific promoters have been identified, particularly for citrus fruit development. RESULTS: In this study, we report five citrus fruit-specific/preferential promoters for genetic engineering. Additionally, we have characterized a novel fruit-preferential promoter from plum. Genes specifically expressed in fruit tissues were selected and their isolated promoter regions were fused with the GUSPlus reporter gene for evaluation in transgenic plants. Stable transformation in Micro-Tom tomato demonstrated that the candidate promoter regions exhibit differing levels of expression and with varying degrees of fruit specificity. CONCLUSIONS: Among the five candidate citrus promoters characterized in this study, the CitSEP promoter showed a fruit-specific expression pattern, while the CitWAX and CitJuSac promoters exhibited high fruit-preferential expression with strong activity in the fruit, weak activity in floral tissues and low or undetectable activity in other tissues. The CitVO1, CitUNK and PamMybA promoters, while exhibiting strong fruit-preferential expression, also showed consistent weak but detectable activity in leaves and other vegetative tissues. Use of these fruit specific/preferential promoters for genetic engineering can help with precise expression of beneficial genes and help with accurate prediction of the activity of new genes in host fruit plants.

Phenotypic Examination of Camelina sativa (L.) Crantz Accessions from the USDA-ARS National Genetics Resource Program.

Camelina sativa (L.) Crntz. is a hardy self-pollinated oilseed plant that belongs to the Brassicaceae family; widely grown throughout the northern hemisphere until the 1940s for production of vegetable oil but was later displaced by higher-yielding rapeseed and sunflower crops. However, interest in camelina as an alternative oil source has been renewed due to its high oil content that is rich in polyunsaturated fatty acids, antioxidants as well as its ability to grow on marginal lands with minimal requirements. For this reason, our group decided to screen the existing (2011) National Genetic Resources Program (NGRP) center collection of camelina for its genetic diversity and provide a phenotypic evaluation of the cultivars available. Properties evaluated include seed and oil traits, developmental and mature morphologies, as well as chromosome content. Selectable marker genes were also evaluated for potential use in biotech manipulation. Data is provided in a raw uncompiled format to allow other researchers to analyze the unbiased information for their own studies. Our evaluation has determined that the NGRP collection has a wide range of genetic potential for both breeding and biotechnological manipulation purposes. Accessions were identified within the NGRP collection that appear to have desirable seed harvest weight (5.06 g/plant) and oil content (44.1%). Other cultivars were identified as having fatty acid characteristics that may be suitable for meal and/or food use, such as low (<2%) erucic acid content, which is often considered for healthy consumption and ranged from a high of 4.79% to a low of 1.83%. Descriptive statistics are provided for a breadth of traits from 41 accessions, as well as raw data, and key seed traits are further explored. Data presented is available for public use.

A bacterial gene codA encoding cytosine deaminase is an effective conditional negative selectable marker in Glycine max.

KEY MESSAGE: Research describes the practical application of the codA negative selection marker in Soybean. Conditions are given for codA selection at both the shooting and rooting stages of regeneration. Conditional negative selection is a powerful technique whereby the absence of a gene product allows survival in otherwise lethal conditions. In plants, the Escherichia coli gene codA has been employed as a negative selection marker. Our research demonstrates that codA can be used as a negative selection marker in soybean, Glycine max. Like most plants, soybean does not contain cytosine deaminase activity and we show here that wild-type seedlings are not affected by inclusion of 5-FC in growth media. In contrast, transgenic G. max plants expressing codA and grown in the presence of more than 200 µg/mL 5-FC exhibit reductions in hypocotyl and taproot lengths, and severe suppression of lateral root development. We also demonstrate a novel negative selection-rooting assay in which codA-expressing aerial tissues or shoot cuttings are inhibited for root formation in media containing 5-FC. Taken together these techniques allow screening during either the regeneration or rooting phase of tissue culture.

AXR1-ECR1 and AXL1-ECR1 heterodimeric RUB-activating enzymes diverge in function in Arabidopsis thaliana.

RELATED TO UBIQUITIN (RUB) modification of CULLIN (CUL) subunits of the CUL-RING ubiquitin E3 ligase (CRL) superfamily regulates CRL ubiquitylation activity. RUB modification requires E1 and E2 enzymes that are analogous to, but distinct from, those activities required for UBIQUITIN (UBQ) attachment. Gene duplications are widespread in angiosperms, and in line with this observation, components of the RUB conjugation pathway are found in multiples in Arabidopsis. To further examine the extent of redundancy within the RUB pathway, we undertook biochemical and genetic characterizations of one such duplication event- the duplication of the genes encoding a subunit of the RUB E1 into AUXIN RESISTANT1 (AXR1) and AXR1-LIKE1 (AXL1). In vitro, the two proteins have similar abilities to function with E1 C-TERMINAL-RELATED1 (ECR1) in catalyzing RUB1 activation and RUB1-ECR1 thioester formation. Using mass spectrometry, endogenous AXR1 and AXL1 proteins were found in complex with 3HA-RUB1, suggesting that AXR1 and AXL1 exist in parallel RUB E1 complexes in Arabidopsis. In contrast, AXR1 and AXL1 differ in ability to correct phenotypic defects in axr1-30, a severe loss-of-function AXR1 mutant, when the respective coding sequences are expressed from the same promoter, suggesting differential in vivo functions. These results suggest that while both proteins function in the RUB pathway and are biochemically similar in RUB-ECR1 thioester formation, they are not functionally equivalent.

Regulation of cullin RING ligases.

The ubiquitin/26S proteasome pathway largely mediates selective proteolysis in the nucleus and cytosol. This pathway catalyzes covalent attachment of ubiquitin (UBQ) to substrate proteins in an E1-E2-E3 cascade. Ubiquitin E3 ligases interact with substrates to catalyze UBQ transfer from E2 to substrate. Within the E3 ligase superfamily, cullin RING ligases (CRLs) are significant in plants because they are linked to hormonal signaling, developmental programs, and environmental responses. Thus, knowledge of CRL regulation is required for a complete understanding of these processes. A major mechanism modulating CRL activity is modification of the cullin subunit by RUB (RELATED TO UBIQUITIN), a ubiquitin-like protein, and demodification by the COP9 signalosome (CSN). CULLIN-ASSOCIATED NEDD8-DISSOCIATED 1 (CAND1) interacts with CRLs, affecting both rubylation and derubylation. Described here are the pathways, regulation, and biological function of rubylation and derubylation, as well as future directions and outstanding questions.

The type III effector repertoire of Pseudomonas syringae pv. syringae B728a and its role in survival and disease on host and non-host plants.

The bacterial plant pathogen Pseudomonas syringae injects a large repertoire of effector proteins into plant cells using a type III secretion apparatus. Effectors can trigger or suppress defences in a host-dependent fashion. Host defences are often accompanied by programmed cell death, while interference with defences is sometimes associated with cell death suppression. We previously predicted the effector repertoire of the sequenced bean pathogen P. syringae pv. syringae (Psy) B728a using bioinformatics. Here we show that PsyB728a is also pathogenic on the model plant species Nicotiana benthamiana (tobacco). We confirm our effector predictions and clone the nearly complete PsyB728a effector repertoire. We find effectors to have different cell death-modulating activities and distinct roles during the infection of the susceptible bean and tobacco hosts. Unexpectedly, we do not find a strict correlation between cell death-eliciting and defence-eliciting activity and between cell death-suppressing activity and defence-interfering activity. Furthermore, we find several effectors with quantitative avirulence activities on their susceptible hosts, but with growth-promoting effects on Arabidopsis thaliana, a species on which PsyB728a does not cause disease. We conclude that P. syringae strains may have evolved large effector repertoires to extend their host ranges or increase their survival on various unrelated plant species.