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Main Conclusion: Specific sequences within the leader intron of a soybean polyubiquitin gene stimulated gene expression when placed either within a synthetic intron or upstream of a core promoter. The intron in the 5' untranslated region of the soybean polyubiquitin promoter, Gmubi, seems to contribute to the high activity of this promoter. To identify the stimulatory sequences within the intron, ten different sequential intronic sequences of 40 nt were isolated, cloned as tetrameric repeats and placed upstream of a minimal cauliflower mosaic virus 35S (35S) core promoter, which was used to control expression of the green fluorescent protein. Intron fragment tetramers were also cloned within a modified, native intron, creating a Synthetic INtron Cassette (SINC), which was then placed downstream of Gmubi and 35S core promoters. Intron fragment tetramers and SINC constructs were evaluated using transient expression in lima bean cotyledons and stable expression in soybean hairy roots. Intron fragments, used as tetramers upstream of the 35S core promoter, yielded up to 80 times higher expression than the core promoter in transient expression analyses and ten times higher expression in stably transformed hairy roots. Tetrameric intronic fragments, cloned downstream of the Gmubi and 35S core promoters and within the synthetic intron, also yielded increased transient and stable GFP expression that was up to 4 times higher than Gmubi alone and up to 40 times higher than the 35S core promoter alone. These intron fragments contain sequences that seem to act as promoter regulatory elements and may contribute to the increased expression observed with this native strong promoter. Intron regulatory elements and synthetic introns may provide additional tools for increasing transgene expression in plants.

Sedentary plant-parasitic cyst nematodes are biotrophs that cause significant losses in agriculture. Parasitism is based on modifications of host root cells that lead to the formation of a hypermetabolic feeding site (a syncytium) from which nematodes withdraw nutrients. The host cell cycle is activated in an initial cell selected by the nematode for feeding, followed by activation of neighboring cells and subsequent expansion of feeding site through fusion of hundreds of cells. It is generally assumed that nematodes manipulate production and signaling of the plant hormone cytokinin to activate cell division. In fact, nematodes have been shown to produce cytokinin in vitro; however, whether the hormone is secreted into host plants and plays a role in parasitism remained unknown. Here, we analyzed the spatiotemporal activation of cytokinin signaling during interaction between the cyst nematode, Heterodera schachtii, and Arabidopsis using cytokinin-responsive promoter:reporter lines. Our results showed that cytokinin signaling is activated not only in the syncytium but also in neighboring cells to be incorporated into syncytium. An analysis of nematode infection on mutants that are deficient in cytokinin or cytokinin signaling revealed a significant decrease in susceptibility of these plants to nematodes. Further, we identified a cytokinin-synthesizing isopentenyltransferase gene in H. schachtii and show that silencing of this gene in nematodes leads to a significant decrease in virulence due to a reduced expansion of feeding sites. Our findings demonstrate the ability of a plant-parasitic nematode to synthesize a functional plant hormone to manipulate the host system and establish a long-term parasitic interaction.

CLE peptides are small extracellular proteins important in regulating plant meristematic activity through the CLE-receptor kinase-WOX signalling module. Stem cell pools in the SAM (shoot apical meristem), RAM (root apical meristem) and vascular cambium are controlled by CLE signalling pathways. Interestingly, plant-parasitic cyst nematodes secrete CLE-like effector proteins, which act as ligand mimics of plant CLE peptides and are required for successful parasitism. Recently, we demonstrated that Arabidopsis CLE receptors CLAVATA1 (CLV1), the CLAVATA2 (CLV2)/CORYNE (CRN) heterodimer receptor complex and RECEPTOR-LIKE PROTEIN KINASE 2 (RPK2), which transmit the CLV3 signal in the SAM, are required for perception of beet cyst nematode Heterodera schachtii CLEs. Reduction in nematode infection was observed in clv1, clv2, crn, rpk2 and combined double and triple mutants. In an effort to develop nematode resistance in an agriculturally important crop, orthologues of Arabidopsis receptors including CLV1, CLV2, CRN and RPK2 were identified from soybean, a host for the soybean cyst nematode Heterodera glycines. For each of the receptors, there are at least two paralogues in the soybean genome. Localization studies showed that most receptors are expressed in the root, but vary in their level of expression and spatial expression patterns. Expression in nematode-induced feeding cells was also confirmed. In vitro direct binding of the soybean receptors with the HgCLE peptide was analysed. Knock-down of the receptors in soybean hairy roots showed enhanced resistance to SCN. Our findings suggest that targeted disruption of nematode CLE signalling may be a potential means to engineer nematode resistance in crop plants., (© 2015 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd.)

Key Message: An extended version of an intron-containing soybean polyubiquitin promoter gave very high levels of gene expression using three different validation tools. The intron-containing Glycine max polyubiquitin promoter (Gmubi) is able to regulate expression levels five times higher than the widely used CaMV35S promoter. In this study, eleven Gmubi derivatives were designed and evaluated to determine which regions contributed to the high levels of gene expression, observed with this promoter. Derivative constructs regulating GFP were evaluated using transient expression in lima bean cotyledons and stable expression in soybean hairy roots. With both expression systems, removal of the intron in the 5'UTR led to reduced levels of gene expression suggesting a role of the intron in promoter activity. Promoter constructs containing an internal intron duplication and upstream translocations of the intron resulted in higher and similar expression levels to Gmubi, respectively, indicating the presence of enhancers within the intron. Evaluation of 5' distal extensions of the Gmubi promoter resulted in significantly higher levels of GFP expression, suggesting the presence of upstream regulatory elements. A twofold increase in promoter strength was obtained when Gmubi was extended 1.5 kb upstream to generate GmubiXL (2.4 kb total length). In stably transformed soybean plants containing GFP regulated by CaMV35S, Gmubi and GmubiXL, the GmubiXL promoter clearly produced the highest levels of gene expression, with especially high GFP fluorescence in the vascular tissue and root tips. Use of GmubiXL leads to very high levels of gene expression in soybean and represents a native soybean promoter, which may be useful for regulating transgene expression for both basic and applied research.

Hosta virus X (HVX) is rapidly becoming a serious pathogen of commercially important hosta plants worldwide. We report here biological and molecular characterization of a U.S. isolate of HVX, HVX-37. HVX-37 infectivity was tested in 21 hosta cultivars over three growth seasons, and three types of responses were defined based upon the ability of the virus to cause local and/or systemic infections. Four cultivars resistant to systemic HVX infection were identified. The full-length sequence of the HVX-37 genome was determined, the first complete sequence of a U.S. HVX isolate. Comparison with the previously sequenced HVX-Korea (Kr) genome revealed a high level of sequence similarity, as well as some differences. Notably, a 105-nucleotide long, near-perfect direct repeat in the Kr isolate is absent in HVX-37. The accuracy of the HVX-37 genome sequence was confirmed by infectivity of in vitro transcripts synthesized from a full-length HVX-37 cDNA on Nicotiana benthamiana and hosta plants.