Jacob O. Brunkard

MicroRNA regulation of plant innate immune receptors

Plant genomes contain large numbers of cell surface leucine-rich repeat (LRR) and intracellular nucleotide binding (NB)-LRR immune receptors encoded by resistance (R) genes that recognize specific pathogen effectors and trigger resistance responses. The unregulated expression of NB-LRR genes can trigger autoimmunity in the absence of pathogen infection and inhibit plant growth. Despite the potential serious consequence on agricultural production, the mechanisms regulating R-gene expression are not well understood. We identified microRNA (miRNA) progenitor genes precursor transcripts, and two miRNAs [nta-miR6019 (22-nt) and nta-miR6020 (21-nt)] that guide cleavage of transcripts of the Toll and Interleukin-1 receptor-NB-LRR immune receptor N from tobacco that confers resistance to tobacco mosaic virus (TMV). We further showed that cleavage by nta-miR6019 triggers RNA-dependent RNA polymerase 6- and ribonuclease Dicer-like 4-dependent biogenesis of 21-nt secondary siRNAs “in phase” with the 22-nt miR6019 cleavage site. Furthermore, we found that processing of the 22-nt nta-miR6019 depended on an asymmetric bulge caused by mismatch in the nta-miR6019 precursor. Interestingly, coexpression of N with nta-miR6019 and nta-miR6020 resulted in attenuation of N-mediated resistance to TMV, indicating that these miRNAs have functional roles in NB-LRR regulation. Using a bioinformatics approach, we identified six additional 22-nt miRNA and two 21-nt miRNA families from three Solanaceae species—tobacco, tomato, and potato. We show that members of these miRNA families cleave transcripts of predicted functional R genes and trigger production of phased secondary 21-nt siRNAs. Our results demonstrate a conserved role for miRNAs and secondary siRNAs in NB-LRR/LRR immune receptor gene regulation and pathogen resistance in Solanaceae.

Redox states of plastids and mitochondria differentially regulate intercellular transport via plasmodesmata

Recent studies suggest that intercellular transport via plasmodesmata (PD) is regulated by cellular redox state. Until now, this relationship has been unclear, as increased production of reactive oxygen species (ROS) has been associated with both increased and decreased intercellular transport via PD. Here, we show that silencing two genes that both increase transport via PD, INCREASED SIZE EXCLUSION LIMIT1 (ISE1) and ISE2, alters organelle redox state. Using redox-sensitive green fluorescent proteins targeted to the mitochondria or plastids, we show that, relative to wild-type leaves, plastids are more reduced in both ISE1- and ISE2-silenced leaves, whereas mitochondria are more oxidized in ISE1-silenced leaves. We further show that PD transport is positively regulated by ROS production in mitochondria following treatment with salicylhydroxamic acid but negatively regulated by an oxidative shift in both chloroplasts and mitochondria following treatment with paraquat. Thus, oxidative shifts in the mitochondrial redox state positively regulate intercellular transport in leaves, but oxidative shifts in the plastid redox state counteract this effect and negatively regulate intercellular transport. This proposed model reconciles previous contradictory evidence relating ROS production to PD transport and supports accumulating evidence that mitochondria and plastids are crucial regulators of PD function.

Organelle–nucleus cross-talk regulates plant intercellular communication via plasmodesmata

We use Arabidopsis thaliana embryogenesis as a model system for studying intercellular transport via plasmodesmata (PD). A forward genetic screen for altered PD transport identified increased size exclusion limit (ise) 1 and ise2 mutants with increased intercellular transport of fluorescent 10-kDa tracers. Both ise1 and ise2 exhibit increased formation of twinned and branched PD. ISE1 encodes a mitochondrial DEAD-box RNA helicase, whereas ISE2 encodes a DEVH-type RNA helicase. Here, we show that ISE2 foci are localized to the chloroplast stroma. Surprisingly, plastid development is defective in both ise1 and ise2 mutant embryos. In an effort to understand how RNA helicases that localize to different organelles have similar impacts on plastid and PD development/function, we performed whole-genome expression analyses. The most significantly affected class of transcripts in both mutants encode products that target to and enable plastid function. These results reinforce the importance of plastid-mitochondria-nucleus cross-talk, add PD as a critical player in the plant cell communication network, and thereby illuminate a previously undescribed signaling pathway dubbed organelle–nucleus-plasmodesmata signaling. Several genes with roles in cell wall synthesis and modification are also differentially expressed in both mutants, providing new targets for investigating PD development and function.

Chloroplasts extend stromules independently and in response to internal redox signals

Significance Chloroplasts are critical, dynamic organelles in plant cells responsible for photosynthesis and myriad other aspects of metabolism. In recent years, plant cell biologists have increasingly focused on the formation of thin, long extensions from plastids called “stromules.” Although stromules have been observed in all land plant species and cell types investigated, we do not know why these projections form or what they do. Here we demonstrate that stromules form in response to light-related redox signals inside the chloroplast. We then show that chloroplasts extracted from plant cells can make stromules independently. These discoveries suggest that stromules may be involved in transmitting signals from within the chloroplast to other subcellular compartments. A fundamental mystery of plant cell biology is the occurrence of “stromules,” stroma-filled tubular extensions from plastids (such as chloroplasts) that are universally observed in plants but whose functions are, in effect, completely unknown. One prevalent hypothesis is that stromules exchange signals or metabolites between plastids and other subcellular compartments, and that stromules are induced during stress. Until now, no signaling mechanisms originating within the plastid have been identified that regulate stromule activity, a critical missing link in this hypothesis. Using confocal and superresolution 3D microscopy, we have shown that stromules form in response to light-sensitive redox signals within the chloroplast. Stromule frequency increased during the day or after treatment with chemicals that produce reactive oxygen species specifically in the chloroplast. Silencing expression of the chloroplast NADPH-dependent thioredoxin reductase, a central hub in chloroplast redox signaling pathways, increased chloroplast stromule frequency, whereas silencing expression of nuclear genes related to plastid genome expression and tetrapyrrole biosynthesis had no impact on stromules. Leucoplasts, which are not photosynthetic, also made more stromules in the daytime. Leucoplasts did not respond to the same redox signaling pathway but instead increased stromule formation when exposed to sucrose, a major product of photosynthesis, although sucrose has no impact on chloroplast stromule frequency. Thus, different types of plastids make stromules in response to distinct signals. Finally, isolated chloroplasts could make stromules independently after extraction from the cytoplasm, suggesting that chloroplast-associated factors are sufficient to generate stromules. These discoveries demonstrate that chloroplasts are remarkably autonomous organelles that alter their stromule frequency in reaction to internal signal transduction pathways.

Plasmodesmata dynamics are coordinated by intracellular signaling pathways.

Membrane-lined channels called plasmodesmata (PD) connect the cytoplasts of adjacent plant cells across the cell wall, permitting intercellular movement of small molecules, proteins, and RNA. Recent genetic screens for mutants with altered PD transport identified genes suggesting that chloroplasts play crucial roles in coordinating PD transport. Complementing this discovery, studies manipulating expression of PD-localized proteins imply that changes in PD transport strongly impact chloroplast biology. Ongoing efforts to find genes that control root and stomatal development reveal the critical role of PD in enforcing tissue patterning, and newly discovered PD-localized proteins show that PD influence development, intracellular signaling, and defense against pathogens. Together, these studies demonstrate that PD function and formation are tightly integrated with plant physiology.

The cytosol must flow: intercellular transport through plasmodesmata.

Plant cells are connected across cell walls by nanoscopic channels called plasmodesmata (PD), which allow plant cells to share resources and exchange signaling molecules. Several protein components of PD membranes have been identified, and recent advances in superresolution live-cell microscopy are illuminating PD ultrastructure. Restricting transport through PD is crucial for morphogenesis, since hormones and hundreds of transcription factors regularly move through PD, and this transport must stop to allow cells to begin differentiating. Chloroplasts and mitochondria regulate PD function through signal transduction networks that coordinate plant physiology and development. Recent discoveries on the relationships of land plants and their algal relatives suggest that PD have evolved independently in several lineages, emphasizing the importance of cytosolic bridges in multicellular biology.

Investigating plasmodesmata genetics with virus-induced gene silencing and an agrobacterium-mediated GFP movement assay.

Plasmodesmata (PD) are channels that connect the cytoplasm of adjacent plant cells, permitting intercellular transport and communication. PD function and formation are essential to plant growth and development, but we still know very little about the genetic pathways regulating PD transport. Here, we present a method for assaying changes in the rate of PD transport following genetic manipulation. Gene expression in leaves is modified by virus-induced gene silencing. Seven to ten days after infection with Tobacco rattle virus carrying a silencing trigger, the gene(s) of interest is silenced in newly arising leaves. In these new leaves, individual cells are then transformed with Agrobacterium to express GFP, and the rate of GFP diffusion via PD is measured. By measuring GFP diffusion both within the epidermis and between the epidermis and mesophyll, the assay can be used to study the effects of silencing a gene(s) on PD transport in general, or transport through secondary PD specifically. Plant biologists working in several fields will find this assay useful, since PD transport impacts plant physiology, development, and defense.

Comment on “A promiscuous intermediate underlies the evolution of LEAFY DNA binding specificity”

Sayou et al. (Reports, 7 February 2014, p. 645) proposed a new model for evolution of transcription factors without gene duplication, using LEAFY as an archetype. Their proposal contradicts the evolutionary history of plants and ignores evidence that LEAFY evolves through gene duplications. Within their data set, we identified a moss with multiple LEAFY orthologs, which contests their model and supports that LEAFY evolves through duplications.

A Two-Headed Monster to Avert Disaster: HBS1/SKI7 Is Alternatively Spliced to Build Eukaryotic RNA Surveillance Complexes

The cytosolic RNA exosome, a 3′→5′ exoribonuclease complex, contributes to mRNA degradation in eukaryotes, limiting the accumulation of poorly-translated, improperly translated, or aberrant mRNA species. Disruption of cytosolic RNA exosome activity allows aberrant RNA species to accumulate, which can then be detected by host antiviral immune systems as a signature of pathogen infection, activating antiviral defenses. SKI7 is a critical component of the cytosolic RNA exosome in yeast, bridging the catalytic exoribonuclease core with the SKI2/SKI3/SKI8 adaptor complex that guides aberrant RNA substrates into the exosome. The ortholog of SKI7 was only recently identified in humans as an alternative splice form of the HBS1 gene, which encodes a decoding factor translational GTPase that rescues stalled ribosomes. Here, we identify the plant orthologs of HBS1/SKI7. We found that HBS1 and SKI7 are typically encoded by alternative splice forms of a single locus, although some plant lineages have evolved subfunctionalized genes that apparently encode only HBS1 or only SKI7. In all plant lineages examined, the SKI7 gene is subject to regulation by alternative splicing that can yield unproductive transcripts, either by removing deeply conserved SKI7 coding sequences, or by introducing premature stop codons that render SKI7 susceptible to nonsense-mediated decay. Taking a comparative, evolutionary approach, we define crucial features of the SKI7 protein shared by all eukaryotes, and use these deeply conserved features to identify SKI7 proteins in invertebrate lineages. We conclude that SKI7 is a conserved cytosolic RNA exosome subunit across eukaryotic lineages, and that SKI7 is consistently regulated by alternative splicing, suggesting broad coordination of nuclear and cytosolic RNA metabolism.

Ties that bind: the integration of plastid signalling pathways in plant cell metabolism

Plastids are critical organelles in plant cells that perform diverse functions and are central to many metabolic pathways. Beyond their major roles in primary metabolism, of which their role in photosynthesis is perhaps best known, plastids contribute to the biosynthesis of phytohormones and other secondary metabolites, store critical biomolecules, and sense a range of environmental stresses. Accordingly, plastid-derived signals coordinate a host of physiological and developmental processes, often by emitting signalling molecules that regulate the expression of nuclear genes. Several excellent recent reviews have provided broad perspectives on plastid signalling pathways. In this review, we will highlight recent advances in our understanding of chloroplast signalling pathways. Our discussion focuses on new discoveries illuminating how chloroplasts determine life and death decisions in cells and on studies elucidating tetrapyrrole biosynthesis signal transduction networks. We will also examine the role of a plastid RNA helicase, ISE2, in chloroplast signalling, and scrutinize intriguing results investigating the potential role of stromules in conducting signals from the chloroplast to other cellular locations.