RGI-GOLVEN signaling promotes cell surface immune receptor abundance to regulate plant immunity.

Plant immune responses must be tightly controlled for proper allocation of resources for growth and development. In plants, endogenous signaling peptides regulate developmental and growth-related processes. Recent research indicates that some of these peptides also have regulatory functions in the control of plant immune responses. This classifies these peptides as phytocytokines as they show analogies with metazoan cytokines. However, the mechanistic basis for phytocytokine-mediated regulation of plant immunity remains largely elusive. Here, we identify GOLVEN2 (GLV2) peptides as phytocytokines in Arabidopsis thaliana. GLV2 signaling enhances sensitivity of plants to elicitation with immunogenic bacterial elicitors and contributes to resistance against virulent bacterial pathogens. GLV2 is perceived by ROOT MERISTEM GROWTH FACTOR 1 INSENSITIVE (RGI) receptors. RGI mutants show reduced elicitor sensitivity and enhanced susceptibility to bacterial infection. RGI3 forms ligand-induced complexes with the pattern recognition receptor (PRR) FLAGELLIN SENSITIVE 2 (FLS2), suggesting that RGIs are part of PRR signaling platforms. GLV2-RGI signaling promotes PRR abundance independent of transcriptional regulation and controls plant immunity via a previously undescribed mechanism of phytocytokine activity.

The HK5 and HK6 cytokinin receptors mediate diverse developmental pathways in rice.

The phytohormone cytokinin regulates diverse aspects of plant growth and development. Our understanding of the metabolism and perception of cytokinin has made great strides in recent years, mostly from studies of the model dicot Arabidopsis Here, we employed a CRISPR/Cas9-based approach to disrupt a subset of cytokinin histidine kinase (HK) receptors in rice (Oryza sativa) in order to explore the role of cytokinin in a monocot species. In hk5 and hk6 single mutants, the root growth, leaf width, inflorescence architecture and/or floral development were affected. The double hk5 hk6 mutant showed more substantial defects, including severely reduced root and shoot growth, a smaller shoot apical meristem, and an enlarged root cap. Flowering was delayed in the hk5 hk6 mutant and the panicle was significantly reduced in size and infertile due to multiple defects in floral development. The hk5 hk6 mutant also exhibited a severely reduced cytokinin response, consistent with the developmental phenotypes arising from a defect in cytokinin signaling. These results indicate that HK5 and HK6 act as cytokinin receptors, with overlapping functions to regulate diverse aspects of rice growth and development.

What a tangled web it weaves: auxin coordination of stem cell maintenance and flower production.

Robust agricultural yields require consistent flower production throughout fluctuating environmental conditions. Floral primordia are produced in the inflorescence meristem, which contains a pool of continuously dividing stem cells. Daughter cells of these divisions either retain stem cell identity or are pushed to the SAM periphery, where they become competent to develop into floral primordia after receiving the appropriate signal. Thus, flower production is inherently linked to regulation of the stem cell pool. The plant hormone auxin promotes flower development throughout its early phases and has been shown to interact with the molecular pathways regulating stem cell maintenance. Here, we will summarize how auxin signaling contributes to stem cell maintenance and promotes flower development through the early phases of initiation, outgrowth, and floral fate establishment. Recent advances in this area suggest that auxin may serve as a signal that integrates stem cell maintenance and new flower production.

A PXY-Mediated Transcriptional Network Integrates Signaling Mechanisms to Control Vascular Development in Arabidopsis.

The cambium and procambium generate the majority of biomass in vascular plants. These meristems constitute a bifacial stem cell population from which xylem and phloem are specified on opposing sides by positional signals. The PHLOEM INTERCALATED WITH XYLEM (PXY) receptor kinase promotes vascular cell division and organization. However, how these functions are specified and integrated is unknown. Here, we mapped a putative PXY-mediated transcriptional regulatory network comprising 690 transcription factor-promoter interactions in Arabidopsis (Arabidopsis thaliana). Among these interactions was a feedforward loop containing transcription factors WUSCHEL HOMEOBOX RELATED14 (WOX14) and TARGET OF MONOPTEROS6 (TMO6), each of which regulates the expression of the gene encoding a third transcription factor, LATERAL ORGAN BOUNDARIES DOMAIN4 (LBD4). PXY signaling in turn regulates the WOX14, TMO6, and LBD4 feedforward loop to control vascular proliferation. Genetic interaction between LBD4 and PXY suggests that LBD4 marks the phloem-procambium boundary, thus defining the shape of the vascular bundle. These data collectively support a mechanism that influences the recruitment of cells into the phloem lineage, and they define the role of PXY signaling in this context in determining the arrangement of vascular tissue.

BAM1/2 receptor kinase signaling drives CLE peptide-mediated formative cell divisions in Arabidopsis roots.

Cell division is often regulated by extracellular signaling networks to ensure correct patterning during development. In Arabidopsis, the SHORT-ROOT (SHR)/SCARECROW (SCR) transcription factor dimer activates CYCLIND6;1 (CYCD6;1) to drive formative divisions during root ground tissue development. Here, we show plasma-membrane-localized BARELY ANY MERISTEM1/2 (BAM1/2) family receptor kinases are required for SHR-dependent formative divisions and CYCD6;1 expression, but not SHR-dependent ground tissue specification. Root-enriched CLE ligands bind the BAM1 extracellular domain and are necessary and sufficient to activate SHR-mediated divisions and CYCD6;1 expression. Correspondingly, BAM-CLE signaling contributes to the restriction of formative divisions to the distal root region. Additionally, genetic analysis reveals that BAM-CLE and SHR converge to regulate additional cell divisions outside of the ground tissues. Our work identifies an extracellular signaling pathway regulating formative root divisions and provides a framework to explore this pathway in patterning and evolution.

Type-B response regulators of rice play key roles in growth, development and cytokinin signaling.

Cytokinins are plant hormones with crucial roles in growth and development. Although cytokinin signaling is well characterized in the model dicot Arabidopsis, we are only beginning to understand its role in monocots, such as rice (Oryza sativa) and other cereals of agronomic importance. Here, we used primarily a CRISPR/Cas9 gene-editing approach to characterize the roles of a key family of transcription factors, the type-B response regulators (RRs), in cytokinin signaling in rice. Results from the analysis of single rr mutants as well as higher-order rr21/22/23 mutant lines revealed functional overlap as well as subfunctionalization within members of the gene family. Mutant phenotypes associated with decreased activity of rice type-B RRs included effects on leaf and root growth, inflorescence architecture, flower development and fertilization, trichome formation and cytokinin sensitivity. Development of the stigma brush involved in pollen capture was compromised in the rr21/22/23 mutant, whereas anther development was compromised in the rr24 mutant. Novel as well as conserved roles for type-B RRs in the growth and development of a monocot compared with dicots were identified.

Control of plant stem cell function by conserved interacting transcriptional regulators.

Plant stem cells in the shoot apical meristem (SAM) and root apical meristem are necessary for postembryonic development of aboveground tissues and roots, respectively, while secondary vascular stem cells sustain vascular development. WUSCHEL (WUS), a homeodomain transcription factor expressed in the rib meristem of the Arabidopsis SAM, is a key regulatory factor controlling SAM stem cell populations, and is thought to establish the shoot stem cell niche through a feedback circuit involving the CLAVATA3 (CLV3) peptide signalling pathway. WUSCHEL-RELATED HOMEOBOX 5 (WOX5), which is specifically expressed in the root quiescent centre, defines quiescent centre identity and functions interchangeably with WUS in the control of shoot and root stem cell niches. WOX4, expressed in Arabidopsis procambial cells, defines the vascular stem cell niche. WUS/WOX family proteins are evolutionarily and functionally conserved throughout the plant kingdom and emerge as key actors in the specification and maintenance of stem cells within all meristems. However, the nature of the genetic regime in stem cell niches that centre on WOX gene function has been elusive, and molecular links underlying conserved WUS/WOX function in stem cell niches remain unknown. Here we demonstrate that the Arabidopsis HAIRY MERISTEM (HAM) family of transcription regulators act as conserved interacting cofactors with WUS/WOX proteins. HAM and WUS share common targets in vivo and their physical interaction is important in driving downstream transcriptional programs and in promoting shoot stem cell proliferation. Differences in the overlapping expression patterns of WOX and HAM family members underlie the formation of diverse stem cell niche locations, and the HAM family is essential for all of these stem cell niches. These findings establish a new framework for the control of stem cell production during plant development.

ZHOUPI and KERBEROS Mediate Embryo/Endosperm Separation by Promoting the Formation of an Extracuticular Sheath at the Embryo Surface.

Arabidopsis thaliana seed development requires the concomitant development of two zygotic compartments, the embryo and the endosperm. Following fertilization, the endosperm expands and the embryo grows invasively through the endosperm, which breaks down. Here, we describe a structure we refer to as the embryo sheath that forms on the surface of the embryo as it starts to elongate. The sheath is deposited outside the embryonic cuticle and incorporates endosperm-derived material rich in extensin-like molecules. Sheath production is dependent upon the activity of ZHOUPI, an endosperm-specific transcription factor necessary for endosperm degradation, embryo growth, embryo-endosperm separation, and normal embryo cuticle formation. We show that the peptide KERBEROS, whose expression is ZHOUPI dependent, is necessary both for the formation of a normal embryo sheath and for embryo-endosperm separation. Finally, we show that the receptor-like kinases GSO1 and GSO2 are required for sheath deposition at the embryo surface but not for production of sheath material in the endosperm. We present a model in which sheath formation depends on the coordinated production of material in the endosperm and signaling within the embryo, highlighting the complex molecular interaction between these two tissues during early seed development.

CLAVATA1 controls distinct signaling outputs that buffer shoot stem cell proliferation through a two-step transcriptional compensation loop.

The regulation of stem cell proliferation in plants is controlled by intercellular signaling pathways driven by the diffusible CLAVATA3 (CLV3p) peptide. CLV3p perception is thought to be mediated by an overlapping array of receptors in the stem cell niche including the transmembrane receptor kinase CLV1, Receptor-Like Protein Kinase 2 (RPK2), and a dimer of the receptor-like protein CLV2 and the CORYNE (CRN) pseudokinase. Mutations in these receptors have qualitatively similar effects on stem cell function but it is unclear if this represents common or divergent signaling outputs. Previous work in heterologous systems has suggested that CLV1, RPK2 and CLV2/CRN could form higher order complexes but it is also unclear what relevance these putative complexes have to in vivo receptor functions. Here I use the in vivo regulation of a specific transcriptional target of CLV1 signaling in Arabidopsis to demonstrate that, despite the phenotypic similarities between the different receptor mutants, CLV1 controls distinct signaling outputs in living stem cell niches independent of other receptors. This regulation is separable from stem cell proliferation driven by WUSCHEL, a proposed common transcriptional target of CLV3p signaling. In addition, in the absence of CLV1, CLV1-related receptor kinases are ectopically expressed but also buffer stem cell proliferation through the auto-repression of their own expression. Collectively these data reveal a unique in vivo role for CLV1 separable from other stem cell receptors and provides a framework for dissecting the signaling outputs in stem cell regulation.

TIR-only protein RBA1 recognizes a pathogen effector to regulate cell death in Arabidopsis.

Detection of pathogens by plants is mediated by intracellular nucleotide-binding site leucine-rich repeat (NLR) receptor proteins. NLR proteins are defined by their stereotypical multidomain structure: an N-terminal Toll-interleukin receptor (TIR) or coiled-coil (CC) domain, a central nucleotide-binding (NB) domain, and a C-terminal leucine-rich repeat (LRR). The plant innate immune system contains a limited NLR repertoire that functions to recognize all potential pathogens. We isolated Response to the bacterial type III effector protein HopBA1 (RBA1), a gene that encodes a TIR-only protein lacking all other canonical NLR domains. RBA1 is sufficient to trigger cell death in response to HopBA1. We generated a crystal structure for HopBA1 and found that it has similarity to a class of proteins that includes esterases, the heme-binding protein ChaN, and an uncharacterized domain of Pasteurella multocida toxin. Self-association, coimmunoprecipitation with HopBA1, and function of RBA1 require two previously identified TIR-TIR dimerization interfaces. Although previously described as distinct in other TIR proteins, in RBA1 neither of these interfaces is sufficient when the other is disrupted. These data suggest that oligomerization of RBA1 is required for function. Our identification of RBA1 demonstrates that "truncated" NLRs can function as pathogen sensors, expanding our understanding of both receptor architecture and the mechanism of activation in the plant immune system.

Tyrosine phosphorylation controls brassinosteroid receptor activation by triggering membrane release of its kinase inhibitor.

Receptor tyrosine kinases control many critical processes in metazoans, but these enzymes appear to be absent in plants. Recently, two Arabidopsis receptor kinases--BRASSINOSTEROID INSENSITIVE 1 (BRI1) and BRI1-ASSOCIATED KINASE1 (BAK1), the receptor and coreceptor for brassinosteroids--were shown to autophosphorylate on tyrosines. However, the cellular roles for tyrosine phosphorylation in plants remain poorly understood. Here, we report that the BRI1 KINASE INHIBITOR 1 (BKI1) is tyrosine phosphorylated in response to brassinosteroid perception. Phosphorylation occurs within a reiterated [KR][KR] membrane targeting motif, releasing BKI1 into the cytosol and enabling formation of an active signaling complex. Our work reveals that tyrosine phosphorylation is a conserved mechanism controlling protein localization in all higher organisms.

Cutting Edge Genetics: CRISPR/Cas9 Editing of Plant Genomes.

The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated nuclease 9 (Cas9) system is a genome editing technology transforming the field of plant biology by virtue of the system's efficiency and specificity. The system has quickly evolved for many diverse applications including multiplex gene mutation, gene replacement and transcriptional control. As CRISPR/Cas9 is increasingly applied to plants, it is becoming clear that each component of the system can be modified to improve editing results. This review aims to highlight common considerations and options when conducting CRISPR/Cas9 experiments.

Plant stem cell maintenance by transcriptional cross-regulation of related receptor kinases.

The CLAVATA3 (CLV3)-CLAVATA1 (CLV1) ligand-receptor kinase pair negatively regulates shoot stem cell proliferation in plants. clv1 null mutants are weaker in phenotype than clv3 mutants, but the clv1 null phenotype is enhanced by mutations in the related receptor kinases BARELY ANY MERISTEM 1, 2 and 3 (BAM1, 2 and 3). The basis of this genetic redundancy is unknown. Here, we demonstrate that the apparent redundancy in the CLV1 clade is in fact due to the transcriptional repression of BAM genes by CLV1 signaling. CLV1 signaling in the rib meristem (RM) of the shoot apical meristem is necessary and sufficient for stem cell regulation. CLV3-CLV1 signaling in the RM represses BAM expression in wild-type Arabidopsis plants. In clv1 mutants, ectopic BAM expression in the RM partially complements the loss of CLV1. BAM regulation by CLV1 is distinct from CLV1 regulation of WUSCHEL, a proposed CLV1 target gene. In addition, quadruple receptor mutants are stronger in phenotype than clv3, pointing to the existence of additional CLV1/BAM ligands. These data provide an explanation for the genetic redundancy seen in the CLV1 clade and reveal a novel feedback operating in the control of plant stem cells.

Somatic hybridization provides segregating populations for the identification of causative mutations in sterile mutants of the moss Physcomitrella patens.

Forward genetics is now straightforward in the moss Physcomitrella patens, and large mutant populations can be screened relatively easily. However, perturbation of development before the formation of gametes currently leaves no route to gene discovery. Somatic hybridization has previously been used to rescue sterile mutants and to assign P. patens mutations to complementation groups, but the cellular basis of the fusion process could not be monitored, and there was no tractable way to identify causative mutations. Here we use fluorescently tagged lines to generate somatic hybrids between Gransden (Gd) and Villersexel (Vx) strains of P. patens, and show that hybridization produces fertile diploid gametophytes that form phenotypically normal tetraploid sporophytes. Quantification of genetic variation between the two parental strains reveals single nucleotide polymorphisms at a frequency of 1/286 bp. Given that the genetic distinction between Gd and Vx strains exceeds that found between pairs of strains that are commonly used for genetic mapping in other plant species, the spore populations derived from hybrid sporophytes provide suitable material for bulk segregant analysis and gene identification by genome sequencing.

The shoot apical meristem regulatory peptide CLV3 does not activate innate immunity.

The Arabidopsis thaliana leucine-rich repeat receptor kinase FLAGELLIN SENSING2 (FLS2) is required for the recognition of bacterial flagellin in innate immunity. Recently, FLS2 was proposed to act as a multispecific receptor recognizing unrelated exogenous and endogenous peptide ligands, including CLAVATA3 (CLV3), a key regulator of shoot meristem stem cell production. Here, we report experimental evidence demonstrating that FLS2 does not recognize CLV3 and that the shoot apical meristem is immune to bacteria independently of CLV3 perception.

A robust methodology to subclassify pseudokinases based on their nucleotide-binding properties.

Protein kinase-like domains that lack conserved residues known to catalyse phosphoryl transfer, termed pseudokinases, have emerged as important signalling domains across all kingdoms of life. Although predicted to function principally as catalysis-independent protein-interaction modules, several pseudokinase domains have been attributed unexpected catalytic functions, often amid controversy. We established a thermal-shift assay as a benchmark technique to define the nucleotide-binding properties of kinase-like domains. Unlike in vitro kinase assays, this assay is insensitive to the presence of minor quantities of contaminating kinases that may otherwise lead to incorrect attribution of catalytic functions to pseudokinases. We demonstrated the utility of this method by classifying 31 diverse pseudokinase domains into four groups: devoid of detectable nucleotide or cation binding; cation-independent nucleotide binding; cation binding; and nucleotide binding enhanced by cations. Whereas nine pseudokinases bound ATP in a divalent cation-dependent manner, over half of those examined did not detectably bind nucleotides, illustrating that pseudokinase domains predominantly function as non-catalytic protein-interaction modules within signalling networks and that only a small subset is potentially catalytically active. We propose that henceforth the thermal-shift assay be adopted as the standard technique for establishing the nucleotide-binding and catalytic potential of kinase-like domains.

An evolutionarily conserved pseudokinase mediates stem cell production in plants.

Sequence comparisons, biochemical experiments, and studies with mutants in transgenic plants show that the Arabidopsis protein CORYNE, currently thought to be a kinase that acts as part of a receptor kinase complex, is likely to be a pseudokinase and not a kinase.

A network of CLAVATA receptors buffers auxin-dependent meristem maintenance.

Plant body plans are elaborated in response to both environmental and endogenous cues. How these inputs intersect to promote growth and development remains poorly understood. During reproductive development, central zone stem cell proliferation in inflorescence meristems is negatively regulated by the CLAVATA3 (CLV3) peptide signalling pathway. In contrast, floral primordia formation on meristem flanks requires the hormone auxin. Here we show that CLV3 signalling is also necessary for auxin-dependent floral primordia generation and that this function is partially masked by both inflorescence fasciation and heat-induced auxin biosynthesis. Stem cell regulation by CLAVATA signalling is separable from primordia formation but is also sensitized to temperature and auxin levels. In addition, we uncover a novel role for the CLV3 receptor CLAVATA1 in auxin-dependent meristem maintenance in cooler environments. As such, CLV3 signalling buffers multiple auxin-dependent shoot processes across divergent thermal environments, with opposing effects on cell proliferation in different meristem regions.