A viral toolbox for conditional and transneuronal gene expression in zebrafish.

The zebrafish is an important model in systems neuroscience but viral tools to dissect the structure and function of neuronal circuitry are not established. We developed methods for efficient gene transfer and retrograde tracing in adult and larval zebrafish by herpes simplex viruses (HSV1). HSV1 was combined with the Gal4/UAS system to target cell types with high spatial, temporal, and molecular specificity. We also established methods for efficient transneuronal tracing by modified rabies viruses in zebrafish. We demonstrate that HSV1 and rabies viruses can be used to visualize and manipulate genetically or anatomically identified neurons within and across different brain areas of adult and larval zebrafish. An expandable library of viruses is provided to express fluorescent proteins, calcium indicators, optogenetic probes, toxins and other molecular tools. This toolbox creates new opportunities to interrogate neuronal circuits in zebrafish through combinations of genetic and viral approaches.

miR393 and secondary siRNAs regulate expression of the TIR1/AFB2 auxin receptor clade and auxin-related development of Arabidopsis leaves.

The phytohormone auxin is a key regulator of plant growth and development that exerts its functions through F-box receptors. Arabidopsis (Arabidopsis thaliana) has four partially redundant of these receptors that comprise the TRANSPORT INHIBITOR RESPONSE1/AUXIN SIGNALING F-BOX1 auxin receptor (TAAR) clade. Recent studies have shown that the microRNA miR393 regulates the expression of different sets of TAAR genes following pathogen infection or nitrate treatment. Here we report that miR393 helps regulate auxin-related development of leaves. We found that AtMIR393B is the predominant source for miR393 in all aerial organs and that miR393 down-regulates all four TAAR genes by guiding the cleavage of their mRNAs. A mutant unable to produce miR393 shows developmental abnormalities of leaves and cotyledons reminiscent of enhanced auxin perception by TAARs. Interestingly, miR393 initiates the biogenesis of secondary siRNAs from the transcripts of at least two of the four TAAR genes. Our results indicate that these siRNAs, which we call siTAARs, help regulate the expression of TAAR genes as well as several unrelated genes by guiding the cleavage of their mRNAs. Thus, miR393 and possibly siTAARs regulate auxin perception and certain auxin-related aspects of leaf development.

Altered expression of an ankyrin-repeat protein results in leaf abnormalities, necrotic lesions, and the elaboration of a systemic signal.

The PR-like proteins, class I beta-1,3-glucanase (GLU I) and chitinase (CHN I), are induced as part of a stereotypic response that can provide protection against viral, bacterial, and fungal pathogens. We have identified two Nicotiana plumbaginifolia ankyrin-repeat proteins, designated Glucanohydrolase Binding Proteins (GBP) 1 and 2, that bind GLU I and CHN I both in vitro and when expressed in yeast cells. Sense as well as antisense transformants of tobacco carrying the GBP1 gene elaborated graft-transmissible acropetally moving signals that induced the downward curling of young leaves. This phenotype was associated with reduced starch, sucrose, and fructose accumulation; the formation of necrotic lesions; and, the induction of markers for the hypersensitive response. GBP1/2 are members of a conserved Plant- Specific Ankyrin- repeat (PANK) family that includes proteins implicated in carbohydrate allocation, reactive oxygen metabolism, hypersensitive cell death, rapid elicitor responses, virus pathogenesis, and auxin signaling. The similarity in phenotype of PANK transformants and transformants altered in carbohydrate metabolism leads us to propose that PANK family members are multifunctional proteins involved in linking plant defense responses and carbohydrate metabolism.