The Arabidopsis Callose Synthase Gene GSL8 Is Required for Cytokinesis and Cell Patterning

Cytokinesis is the division of the cytoplasm and its separation into two daughter cells. Cell plate growth and cytokinesis appear to require callose, but direct functional evidence is still lacking. To determine the role of callose and its synthesis during cytokinesis, we identified and characterized mutants in many members of the GLUCAN SYNTHASE-LIKE (GSL; or CALLOSE SYNTHASE) gene family in Arabidopsis (Arabidopsis thaliana). Most gsl mutants (gsl1–gsl7, gsl9, gsl11, and gsl12) exhibited roughly normal seedling growth and development. However, mutations in GSL8, which were previously reported to be gametophytic lethal, were found to produce seedlings with pleiotropic defects during embryogenesis and early vegetative growth. We found cell wall stubs, two nuclei in one cell, and other defects in cell division in homozygous gsl8 insertional alleles. In addition, gsl8 mutants and inducible RNA interference lines of GSL8 showed reduced callose deposition at cell plates and/or new cell walls. Together, these data show that the GSL8 gene encodes a putative callose synthase required for cytokinesis and seedling maturation. In addition, gsl8 mutants disrupt cellular and tissue-level patterning, as shown by the presence of clusters of stomata in direct contact and by islands of excessive cell proliferation in the developing epidermis. Thus, GSL8 is required for patterning as well as cytokinesis during Arabidopsis development.Cytokinesis divides the cytoplasm of a plant cell by the deposition of plasma membrane and a cell wall during late mitosis. This process requires the phragmoplast, a dynamic, plant-specific cytoskeletal and membranous array, which delivers vesicles containing lipids, proteins, and cell wall components to the division plane to construct the cell plate. Cell plate formation involves several stages: initiation through vesicle fusion, the formation of a tubular-vesicular network, a transition to a solely tubular phase, and then further fusion to form a fenestrated sheet (Samuels et al., 1995). The outward growth of the cell plate leads to its fusion with the parental cell wall (Jürgens, 2005a, 2005b; Backues et al., 2007).Key regulators of cytokinesis include KNOLLE, KEULE, KORRIGAN, and HINKEL, which when defective induce pleiotropic phenotypes and seedling lethality (Lukowitz et al., 1996; Nicol et al., 1998; Zuo et al., 2000; Assaad et al., 2001; Strompen et al., 2002). KNOLLE, a syntaxin homolog, is required for the fusion of exocytic vesicles via a SNARE/SNAP33 complex (Lukowitz et al., 1996; Heese et al., 2001). KEULE, a homolog of yeast Sec1p, regulates syntaxin function by interacting with KNOLLE (Waizenegger et al., 2000; Assaad et al., 2001). KORRIGAN is an endo-1,4-β-glucanase required for cell wall biogenesis during cytokinesis (Zuo et al., 2000). And HINKEL is a kinesin-related protein required for the reorganization of phragmoplast microtubules during cytokinesis (Strompen et al., 2002).Additional regulators include Formin5, TWO-IN-ONE (TIO), and Arabidopsis (Arabidopsis thaliana) dynamin-like proteins (ADLs; Kang et al., 2001, 2003; Hong et al., 2003; Collings et al., 2005; Ingouff et al., 2005; Oh et al., 2005). Formin5 localizes to the cell plate and is an actin-organizing protein involved in cytokinesis and cell polarity. TIO, a Ser/Thr protein kinase, functions in cytokinesis in plant meristems and in gametogenesis (Oh et al., 2005). Members of the Arabidopsis DRP family associate with the developing cell plate, whereas DRP1a (ADL1A) locally constricts tubular membranes, interacts with callose synthase, and may facilitate callose deposition into the lumen.Callose, a β-1,3-glucan polymer with β-1,6-branches (Stone and Clarke, 1992), is synthesized in both sporophytic and gametophytic tissues and appears to play various roles. Callose accumulates at the cell plate during cytokinesis, in plasmodesmata, where it regulates cell-to-cell communication, and in dormant phloem, where it seals sieve plates after mechanical injury, pathogen attack, and metal toxicity (Stone and Clarke, 1992; Samuels et al., 1995; Lucas and Lee, 2004).Twelve GLUCAN SYNYHASE-LIKE (GSL) genes (also known as CALLOSE SYNTHASE [CalS]) have been identified in the Arabidopsis genome based on sequence homology (Richmond and Somerville, 2000; Hong et al., 2001; Enns et al., 2005). A GSL that functions in callose deposition after injury and pathogen treatment is GSL5 (Jacobs et al., 2003). Five other members of the Arabidopsis GSL family are required for microgametogenesis. GSL1 and GSL5 act redundantly to produce a callosic wall that prevents microspore degeneration, and both are needed for fertilization (Enns et al., 2005). GSL2 is required for the callosic wall around pollen mother cells, for the patterning of the pollen exine (Dong et al., 2005), and for callose deposition in the wall and plugs of pollen tubes (Nishikawa et al., 2005). GSL8 and GSL10 are independently required for the asymmetric division of microspores and for the entry of microspores into mitosis (Töller et al., 2008; Huang et al., 2009).Callose is a major component of the cell plate, especially during later plate development (Kakimoto and Shibaoka, 1992; Samuels et al., 1995; Hong et al., 2001). Callose appears to structurally reinforce the developing cell plate after the breakdown of the phragmoplast microtubule array and during plate consolidation (Samuels and Staehelin, 1996; Rensing et al., 2002). It is likely that callose is synthesized at the cell plate rather than in the endoplasmic reticulum and in the Golgi (Kakimoto and Shibaoka, 1988). GSL6 (CalS1) appears to be involved in callose synthesis at the cell plate, since a 35S∷GFP-GSL6 fusion in transgenic BY-2 tobacco (Nicotiana tabacum) cells increases callose accumulation, and GFP fluorescence was found specifically at the cell plate (Hong et al., 2001). However, functional and genetic data on the role of any GSL in Arabidopsis sporophytic cytokinesis are still lacking.Here, we report that GSL8 (CalS10) is required for normal cytokinesis. In addition, gsl8 mutants exhibit excessive cell proliferation and abnormal cell patterning, phenotypes not previously reported for cytokinesis-defective mutants.     

拟南芥编码亮氨酸拉链蛋白的AS2基因在叶发育中具有控制极性建立的作用

在拟南芥 (Arabidopsisthaliana (L .)Heynh .)叶发育研究中 ,as2是一个经典突变体。as2典型的表型是叶片开裂或形成一种小叶状结构。遗传学和分子生物学实验证明 ,AS2基因具有抑制KNOX基因在叶中表达的功能。在本文中 ,我们着重研究了新得到的在Landsbergerecta (Ler)遗传背景下的as2突变体。除了前人报道过的as2表型外 ,新as2突变体的部分叶柄长在叶片的下方 ,形成一种荷叶状结构 ,更严重的甚至长成花丝状叶结构。这两种结构都反映了不正常的叶腹背轴极性分化。在我们所收集到的as2等位突变体中 ,只有在Ler背景下这两种结构才以高频率出现。我们通过图位克隆方法分离了AS2基因。该基因编码一个含有亮氨酸拉链结构的蛋白。在拟南芥中 ,AS2同源基因共 4 3个 ,除AS2外 ,其他基因的功能都不清楚。AS2在叶和花中表达 ,在茎中无表达 ,这种表达模式和as2突变体的表型是吻合的。     

拟南芥编码亮氨酸拉链蛋白的AS2基因在叶发育中具有控制极性建立的作用

在拟南芥(Arabidopsis thaliana (L.) Heynh.)叶发育研究中,as2是一个经典突变体.as2典型的表型是叶片开裂或形成一种小叶状结构.遗传学和分子生物学实验证明,AS2基因具有抑制KNOX基因在叶中表达的功能.在本文中,我们着重研究了新得到的在Landsberg erecta (Ler)遗传背景下的as2突变体.除了前人报道过的as2表型外,新as2突变体的部分叶柄长在叶片的下方,形成一种荷叶状结构,更严重的甚至长成花丝状叶结构.这两种结构都反映了不正常的叶腹背轴极性分化.在我们所收集到的as2等位突变体中,只有在Ler背景下这两种结构才以高频率出现.我们通过图位克隆方法分离了AS2基因.该基因编码一个含有亮氨酸拉链结构的蛋白.在拟南芥中,AS2同源基因共43个,除AS2外,其他基因的功能都不清楚.AS2在叶和花中表达,在茎中无表达,这种表达模式和as2突变体的表型是吻合的.     

拟南芥ASL25/LBD28基因过量表达对叶片形态建成的影响

为研究ASL25/LBD28基因在植物发育过程中的作用,该研究构建了拟南芥ASL25/LBD28的过量表达载体并将其转入野生型拟南芥中,结果发现,ASL25/LBD28基因的过量表达可导致转基因拟南芥的叶片变得狭长;在叶极性发育突变体as2中,ASL25/LBD28基因过量表达导致部分转基因植株在形成1～3片畸形叶后顶端分生组织的发育会终止;而许多转基因植株则会形成许多"针状"叶.扫描电镜观察表明,不正常的叶片近轴面或"针状"叶的表皮细胞具有远轴面化的长条形细胞,说明在as2突变体中过量表达ASL25/LBD28基因影响叶片的极性发育.     

Drosophila convoluted/dALS Is an Essential Gene Required for Tracheal Tube Morphogenesis and Apical Matrix Organization

Insulin-like growth factors (IGFs) control cell and organism growth through evolutionarily conserved signaling pathways. The mammalian acid-labile subunit (ALS) is a secreted protein that complexes with IGFs to modulate their activity. Recent work has shown that a Drosophila homolog of ALS, dALS, can also complex with and modulate the activity of a Drosophila IGF. Here we report the first mutations in the gene encoding dALS. Unexpectedly, we find that these mutations are allelic to a previously described mutation in convoluted (conv), a gene required for epithelial morphogenesis. In conv mutants, the tubes of the Drosophila tracheal system become abnormally elongated without altering tracheal cell number. conv null mutations cause larval lethality, but do not disrupt several processes required for tracheal tube size control, including septate junction formation, deposition of a lumenal/apical extracellular matrix, and lumenal secretion of Vermiform and Serpentine, two putative matrix-modifying proteins. Clearance of lumenal matrix and subcellular localization of clathrin also appear normal in conv mutants. However, we show that Conv/dALS is required for the dynamic organization of the transient lumenal matrix and normal structure of the cuticle that lines the tracheal lumen. These and other data suggest that the Conv/dALS-dependent tube size control mechanism is distinct from other known processes involved in tracheal tube size regulation. Moreover, we present evidence indicating that Conv/dALS has a novel, IGF-signaling independent function in tracheal morphogenesis.     

Nubp1 Is Required for Lung Branching Morphogenesis and Distal Progenitor Cell Survival in Mice

The lung is a complex system in biology and medicine alike. Whereas there is a good understanding of the anatomy and histology of the embryonic and adult lung, less is known about the molecular details and the cellular pathways that ultimately orchestrate lung formation and affect its health. From a forward genetic approach to identify novel genes involved in lung formation, we identified a mutated Nubp1 gene, which leads to syndactyly, eye cataract and lung hypoplasia. In the lung, Nubp1 is expressed in progenitor cells of the distal epithelium. Nubp1(m1Nisw) mutants show increased apoptosis accompanied by a loss of the distal progenitor markers Sftpc, Sox9 and Foxp2. In addition, Nubp1 mutation disrupts localization of the polarity protein Par3 and the mitosis relevant protein Numb. Using knock-down studies in lung epithelial cells, we also demonstrate a function of Nubp1 in regulating centrosome dynamics and microtubule organization. Together, Nubp1 represents an essential protein for lung progenitor survival by coordinating vital cellular processes including cell polarity and centrosomal dynamics.     

The Caenorhabditis Elegans Spe-5 Gene Is Required for Morphogenesis of a Sperm-Specific Organelle and Is Associated with an Inherent Cold-Sensitive Phenotype

The nonrandom segregation of organelles to the appropriate compartment during asymmetric cellular division is observed in many developing systems. Caenorhabditis elegans spermatogenesis is an excellent system to address this issue genetically. The proper progression of spermatogenesis requires specialized intracellular organelles, the fibrous body-membranous organelle complexes (FB-MOs). The FB-MOs play a critical role in cytoplasmic partitioning during the asymmetric cellular division associated with sperm meiosis II. Here we show that spe-5 mutants contain defective, vacuolated FB-MOs and usually arrest spermatogenesis at the spermatocyte stage. Occasionally, spe-5 mutants containing defective FB-MOs will form spermatids that are capable of differentiating into functional spermatozoa. These spe-5 spermatids exhibit an incomplete penetrance for tubulin mis-segregation during the second meiotic division. In addition to morphological and FB-MO segregation defects, all six spe-5 mutants are cold-sensitive, exhibiting a more penetrant sterile phenotype at 16° than 25°. This cold sensitivity could be an inherent property of FB-MO morphogenesis.     

A Novel Gene,ROA, Is Required for Normal Morphogenesis and Discharge of Ascospores in Gibberella zeae

Head blight, caused by Gibberella zeae, is a significant disease among cereal crops, including wheat, barley, and rice, due to contamination of grain with mycotoxins. G. zeae is spread by ascospores forcibly discharged from sexual fruiting bodies forming on crop residues. In this study, we characterized a novel gene, ROA, which is required for normal sexual development. Deletion of ROA (Δroa) resulted in an abnormal size and shape of asci and ascospores but did not affect vegetative growth. The Δroa mutation triggered round ascospores and insufficient cell division after spore delimitation. The asci of the Δroa strain discharged fewer ascospores from the perithecia but achieved a greater dispersal distance than those of the wild-type strain. Turgor pressure within the asci was calculated through the analysis of osmolytes in the epiplasmic fluid. Deletion of the ROA gene appeared to increase turgor pressure in the mutant asci. The higher turgor pressure of the Δroa mutant asci and the mutant spore shape contributed to the longer distance dispersal. When the Δroa mutant was outcrossed with a Δmat1-2 mutant, a strain that contains a green fluorescence protein (GFP) marker in place of the MAT1-2 gene, unusual phenotypic segregation occurred. The ratio of GFP to non-GFP segregation was 1:1; however, all eight spores had the same shape. Taken together, the results of this study suggest that ROA plays multiple roles in maintaining the proper morphology and discharge of ascospores in G. zeae.Gibberella zeae (anamorph: Fusarium graminearum) causes Fusarium head blight in wheat, barley, and rice, as well as ear rot and stalk rot in maize (20, 23). The infected grains are frequently contaminated by mycotoxins, such as trichothecenes and zearalenone, which are harmful to humans and animals (6). The fungus overwinters in crop debris in the form of storage hyphae and develops ephemeral fruiting bodies (perithecia) at warmer temperatures. Ascospores formed within the perithecia are forcibly discharged into the air and are believed to serve as the primary inoculum of the disease (7, 27, 37, 39,–42). Therefore, sexual development and ascospore discharge are important factors in fungal survival and disease initiation.In fungi of the phylum Ascomycota, the sexual cycle is initiated when two genetically distinct nuclei combine to form a binucleate cell (31). As a homothallic fungus, G. zeae possesses the two mating type genes MAT1-1 and MAT1-2 in the haploid genome and therefore does not require a mating partner for sexual development (22, 46). Perithecium initials give rise to small, coiled initials that develop into perithecia filled with asci, tubular sacs of ascospores, which are the products of meiosis. Mature asci extend through the ostiole of perithecia and discharge their ascospores (40).Unique features of cell differentiation are involved in ascus and ascospore morphogenesis. Ascospore delimitation within the ascus and the development of a cell wall between the ascus and ascospore membranes are unique features of the process (31). Most studies of morphogenesis have described these changes in detail; however, much of these data have been limited to microscopic observations. Several genes involved in ascospore morphogenesis have been identified in Neurospora crassa (30), but the detailed mechanisms and genes involved in ascus and ascospore morphogenesis remain to be elucidated. The Round spore (R) mutant of N. crassa was shown to have round ascospores (24), and the gene responsible for this phenotype, rsp, was subsequently cloned (28). However, in G. zeae, no genes have been identified that are involved in ascus and ascospore morphogenesis.Although recent research has shed light on the physiological basis of ascospore discharge, the genetic basis remains largely unknown (38). The main force responsible for the observed shooting is turgor pressure within the extended asci. In G. zeae, a buildup of K⁺ and Cl⁻ ions drives the influx of water and causes turgor pressure that stretches the asci (41). Asci can accumulate polyols as well as ions. In a previous study, it was shown that the polyols are comprised mainly of mannitol and glucose; however, the concentration of these polyols is too low to make a significant contribution to turgor pressure (42). When the turgor pressure exceeds the threshold of the asci, apical pores rupture and ascospores are forcibly discharged (38). Trail et al. (41) estimated that the acceleration of ascospores in G. zeae is 8,500,000 m s⁻² using an iterative model to predict initial velocity. Recently, Yafetto et al. (44) used high-speed video photography to examine several large-spore fungi, including Ascobolus immerses, and to predict acceleration during dispersal. The asci of A. immerses are more than 12-fold larger in diameter than the asci of G. zeae (38). The size difference between these fungi greatly affects the behavior of their projectiles and results in an initial speed for G. zeae that is too great for application of the video photography method (for further discussion, see the supplemental material).To date, only one gene from G. zeae, the calcium ion channel gene cch1, has been shown to be involved in ascospore discharge (12). Deletion of this gene was shown to arrest ascospore discharge without affecting spore and ascus morphology. Since the genomic sequence of G. zeae is now available, the functional analysis of genes involved in sexual development has been accelerated. Random insertional mutagenesis is one strategy that has been used to identify novel genes associated with sexual development (13, 34). Previously, we produced a collection of more than 20,000 mutants from G. zeae by using the restriction enzyme-mediated integration (REMI) transformation procedure (13). In this study, the G. zeae mutant Z43R9901, which was isolated from a screening of REMI transformants, showed an unusual phenotype during sexual development. Further analysis demonstrated that the novel gene ROA is involved in ascospore morphogenesis and discharge in G. zeae. The results of this study increase our understanding of sexual development in the fungus.     

拟南芥温度诱导脂质运载蛋白TIL1参与雌配子体发育

雌配子体的正常发育是种子形成的前提条件之一,拟南芥温度诱导的脂质运载蛋白编码基因TIL1突变使胚珠败育,结实率下降明显。基因表达分析表明T-DNA插入使得TIL1基因敲除,突变体TIL1基因功能缺失;互交实验、Alexander染色、花粉离体培养和胚珠透明实验结果表明till-1突变体雄配子体发育正常、雌配子体胚囊发育有缺陷;通过遗传互补实验证明外源克隆的TIL1基因能恢复突变体的败育表型,并确定了TIL1基因主要在胚珠的胚囊中表达。实验结果表明TIL1基因参与了植物雌配子体发育这一重要的生理过程。     

BAF200 Is Required for Heart Morphogenesis and Coronary Artery Development

ATP-dependent SWI/SNF chromatin remodeling complexes utilize ATP hydrolysis to non-covalently change nucleosome-DNA interactions and are essential in stem cell development, organogenesis, and tumorigenesis. Biochemical studies show that SWI/SNF in mammalian cells can be divided into two subcomplexes BAF and PBAF based on the subunit composition. ARID2 or BAF200 has been defined as an intrinsic subunit of PBAF complex. However, the function of BAF200 in vivo is not clear. To dissect the possible role of BAF200 in regulating embryogenesis and organ development, we generated BAF200 mutant mice and found they were embryonic lethal. BAF200 mutant embryos exhibited multiple cardiac defects including thin myocardium, ventricular septum defect, common atrioventricular valve, and double outlet right ventricle around E14.5. Moreover, we also detected reduced intramyocardial coronary arteries in BAF200 mutants, suggesting that BAF200 is required for proper migration and differentiation of subepicardial venous cells into arterial endothelial cells. Our work revealed that PBAF complex plays a critical role in heart morphogenesis and coronary artery angiogenesis.     

The BRCT Domain of PARP-1 Is Required for Immunoglobulin Gene Conversion

Genetic variation at immunoglobulin (Ig) gene variable regions in B-cells is created through a multi-step process involving deamination of cytosine bases by activation-induced cytidine deaminase (AID) and their subsequent mutagenic repair. To protect the genome from dangerous, potentially oncogenic effects of off-target mutations, both AID activity and mutagenic repair are targeted specifically to the Ig genes. However, the mechanisms of targeting are unknown and recent data have highlighted the role of regulating mutagenic repair to limit the accumulation of somatic mutations resulting from the more widely distributed AID-induced lesions to the Ig genes. Here we investigated the role of the DNA damage sensor poly-(ADPribose)-polymerase-1 (PARP-1) in the repair of AID-induced DNA lesions. We show through sequencing of the diversifying Ig genes in PARP-1^−/− DT40 B-cells that PARP-1 deficiency results in a marked reduction in gene conversion events and enhanced high-fidelity repair of AID-induced lesions at both Ig heavy and light chains. To further characterize the role of PARP-1 in the mutagenic repair of AID-induced lesions, we performed functional analyses comparing the role of engineered PARP-1 variants in high-fidelity repair of DNA damage induced by methyl methane sulfonate (MMS) and the mutagenic repair of lesions at the Ig genes induced by AID. This revealed a requirement for the previously uncharacterized BRCT domain of PARP-1 to reconstitute both gene conversion and a normal rate of somatic mutation at Ig genes, while being dispensable for the high-fidelity base excision repair. From these data we conclude that the BRCT domain of PARP-1 is required to initiate a significant proportion of the mutagenic repair specific to diversifying antibody genes. This role is distinct from the known roles of PARP-1 in high-fidelity DNA repair, suggesting that the PARP-1 BRCT domain has a specialized role in assembling mutagenic DNA repair complexes involved in antibody diversification.     

The Arabidopsis ZED1-Related Kinase Genomic Cluster Is Specifically Required for Effector-Triggered Immunity

CRISPR/Cas9-mediated deletion of an Arabidopsis gene cluster encoding eight kinases supports their immunity-specific roles in sensing pathogenic effectors.

Dear Editor,ZED1-related kinases (ZRKs) associate with the nucleotide binding, Leu-rich repeat (NLR) protein HOPZ-ACTIVATED RESISTANCE1 (ZAR1) to mediate effector-triggered immunity (ETI) against at least three distinct families of pathogenic effector proteins. However, it is unknown whether ZRKs specifically function in ETI or whether they also have additional roles in immunity and/or development. Eight ZRKs are clustered in the Arabidopsis (Arabidopsis thaliana) genome, including the three members with known roles in ETI. Here, we show that an ∼14-kb CRISPR-mediated deletion of the Arabidopsis ZRK genomic cluster specifically affects ETI, with no apparent defects in pattern-recognition-receptor–triggered immunity (PTI) or development.Phytopathogens deliver effector proteins into plant cells that suppress PTI and promote the infection process (Jones and Dangl, 2006). In turn, plants have evolved NLRs that recognize effectors, leading to an ETI response. This recognition often occurs indirectly, whereby NLRs monitor host “sensor” proteins for effector-induced perturbations (Khan et al., 2016). In the absence of their respective NLRs, some of these sensors are effector virulence targets that modulate immunity and development, while others appear to be decoys that mimic virulence targets, with ETI-specific roles (van der Hoorn and Kamoun, 2008; Khan et al., 2018).The ZAR1 NLR recognizes at least six type-III secreted effector (T3SE) families from bacterial phytopathogens. This remarkable immunodiversity appears to be conveyed through associations with members of the receptor-like cytoplasmic kinase XII-2 (RLCK XII-2) family, which all display characteristics of atypical kinases (Lewis et al., 2013; Roux et al., 2014). The ZAR1-mediated ETI responses against the Pseudomonas syringae T3SEs HopZ1a and HopF1r (formerly HopF2a) require ZED1 and ZRK3, whereas recognition of the Xanthomonas campestris T3SE AvrAC requires ZRK1/RKS1 (Lewis et al., 2013; Wang et al., 2015; Seto et al., 2017). ZRKs currently have no ascribed functions outside of ZAR1-associated ETI responses and are therefore considered decoy sensors or adaptors (Lewis et al., 2013; Wang et al., 2015; Khan et al., 2018). However, functional redundancy may exist among members of the ZRK family, masking phenotypes of individual mutants beyond gene-for-gene–type ETI responses (Lewis et al., 2013). We therefore utilized the CRISPR/Cas9 system to knock out the Arabidopsis genomic region containing eight of the 13 members of the RLCK XII-2, including all ZRK genes known to contribute to ETI, to investigate any non-ETI roles of ZRKs. The 14-kb ZRK gene cluster in Arabidopsis Col-0 plants includes ZRK1, ZRK2, ZRK3, ZRK4, ZED1, ZRK6, ZRK7, and ZRK10. A CRISPR/Cas9-mediated deletion of 13.3 kb was accomplished by designing guide RNAs flanking the ends of the ZRK gene cluster, which would result in a double-stranded break on both sides of the ZRK cluster, leaving only the 5′ end 63 nucleotides (21 amino acids) of ZRK10 and the 3′ end 118 nucleotides (39 amino acids) of ZRK7 (Fig. 1A). We obtained a T1 individual (zrk_1.11) homozygous for the deletion, as well as a T1 individual heterozygous for the mutation (zrk_1.10; Fig. 1B), from which we obtained homozygous T2 (zrk_2.11) and T3 (zrk_3.10) plants, respectively. Sequencing results from zrk_2.11 confirmed that the expected region had been deleted (Supplemental Fig. S1). Plants homozygous for the ZRK gene cluster deletion were morphologically indistinguishable from wild-type Col-0 plants, as well as zar1-1 plants (Fig. 1C). In addition, zrk plant fresh weight did not significantly differ from Col-0 plants (Supplemental Fig. S2), indicating that the ZRK cluster does not play a major role in vegetative plant development.Open in a separate windowFigure 1.Deletion of the ZRK gene cluster results in loss of ZRK-mediated ETI and does not significantly alter vegetative growth. A, Representation of ZRK gene cluster before (top) and after (bottom) CRISPR/Cas9-mediated deletion depicting guide RNAs and primers used for genotyping (see Supplemental Methods S1). ZRK KO primers (magenta) were used to confirm the deletion of the ZRK cluster, while ZRK3 primers (green) were used to check if the ZRK cluster was still present in T1 individuals. B, PCR genotyping for deletion of ZRK gene cluster. Amplification of product by ZRK3 F + R primers indicates lack of deletion; amplification by ZRK KO F + R indicates deletion has occurred. Examples for wild type (WT), heterozygous (HT; zrk_1.10), and homozygous for the deletion (HM KO; zrk_1.11) are shown. T1 lines (zrk_1.10 and zrk_1.11) are compared to wild-type Col-0. C, Uninfected morphology of homozygous zrk KO plants (zrk_3.10 or zrk_2.11) compared to Col-0 and zar1-1 plants. Bar = 1 cm. D, Phenotypes of zrk_2.11 plants 7 d after being sprayed with PtoDC3000(hopZ1a; left) or PtoDC3000(hopF1r; right) relative to wild-type Col-0 and zar1-1 plants. Plant immunity and disease image-based quantification of disease symptoms is presented in Supplemental Figure S3A (Laflamme et al., 2016).Next, we wanted to confirm that the deletion of the ZRK gene cluster compromised ZRK-mediated ETI responses. We sprayed the zrk_2.11 line with PtoDC3000(hopZ1a) or PtoDC3000(hopF1r), as both T3SEs require a ZRK as well as the NLR ZAR1 for their recognition in Arabidopsis (Lewis et al., 2013; Seto et al., 2017). We observed that the zrk_2.11 line was susceptible to both PtoDC3000(hopZ1a) and PtoDC3000(hopF1r), and this susceptibility was to the same level as zar1-1 plants as quantified by plant immunity and disease image-based quantification (Fig. 1D, Supplemental Fig. S3, A and C; Laflamme et al., 2016). We observed a similar phenotype for the zrk_3.10 line, confirming that the ZRK cluster deletion compromised ZRK-mediated ETI responses (Supplemental Fig. S3, B and C). Furthermore, the ZAR1-mediated ETI responses against the P. syringae T3SEs HopBA1a, HopX1i, and HopO1c were also lost in zrk_2.11, demonstrating the ZRK-dependence of these ETI responses (Supplemental Fig. S4; Laflamme et al., 2020). To ensure that the ZRK gene cluster deletion specifically impacted ZRK-related ETI responses, the zrk_3.10 and zrk_2.11 lines were also sprayed with PtoDC3000(avrRpt2), an ETI elicitor that does not require a ZRK or ZAR1 for its recognition (Mackey et al., 2003). zrk_3.10 and zrk_2.11 plants remained resistant to PtoDC3000(avrRpt2), indicating that the ZRK gene cluster deletion specifically impacts ZRK-mediated ETI responses (Supplemental Fig. S3C). In addition, growth of virulent PtoDC3000 on the zrk_3.10 and zrk_2.11 lines was unchanged compared to wild-type Col-0 plants, indicating that the ZRKs within this cluster likely do not represent virulence targets (Supplemental Fig. S5).We then examined whether knocking out the ZRK gene cluster impacted PTI. We first measured induction of peroxidase (POX) enzyme activity, as POX enzymes are produced in response to PTI (Mott et al., 2018). After treatment with the PTI elicitor flg22, addition of the POX substrate 5-aminosalicylic acid produces a brown end-product in the presence of active POX enzymes, which is quantified by reading at an optical density of 550 nm (OD₅₅₀; Mott et al., 2018). Twenty h after leaf discs were treated with flg22, zrk_3.10 and zrk_2.11 plants showed the same level of PTI-associated POX activity as wild-type Col-0 plants (Fig. 2A). To further examine the role of the ZRK gene cluster in PTI, we quantified the growth of PtoDC3000ΔhrcC, which is defective in T3SE secretion and is sensitive to altered host PTI responses under high humidity conditions such as those used in our growth assays (Guo et al., 2009; Xin et al., 2016). Growth of PtoDC3000ΔhrcC on zrk_3.10 and zrk_2.11 plants was not significantly different compared to wild-type Col-0 plants (Fig. 2B). In addition, we monitored reactive oxygen species (ROS) production and found that zrk_3.10 and zrk_2.11 plants did not show a significant difference in the ROS burst observed in wild-type Col-0 plants (Fig. 2, C and D). Finally, we treated seedlings with flg22, and found that growth of zrk_3.10 and zrk_2.11 seedlings was inhibited by the same amount as in wild-type Col-0 seedlings, indicative of a similar induction of PTI responses (Fig. 2, E and F; Gómez-Gómez et al., 1999). Together, these results indicate that the ZRK gene cluster does not play a significant role in Arabidopsis PTI responses.Open in a separate windowFigure 2.Deletion of the ZRK gene cluster does not alter pattern-recognition-receptor–triggered immune responses. A, Response to the PTI elicitor flg22 measured by POX activity. Activity from leaf discs was quantified 20 h after treatment with 1 μm of flg22 at a measurement of OD₅₅₀ (n = 6; Mott et al., 2018). B, Bacterial growth of the T3SS-compromised PtoDC3000ΔhrcC on zrk KO plants (zrk_3.10 and zrk_2.11) relative to wild-type Columbia-0 (wild-type Col-0) and zar1-1 plants 3-d postinoculation. Plants were domed for the duration of the experiment (n = 8). C, Response of Col-0, zrk KO plants (zrk_3.10 and zrk_2.11), and fls2 to the PTI elicitor flg22 measured using luminol-based detection of ROS over a time course of 60 min, with relative light units measured every 2 min (n = 12). D, Boxplots of total relative light units over a period of 30 min from treatments in C (n = 12). E, Growth inhibition of seedlings 7 d after treatment with 1 μm of flg22. F, Seedling growth inhibition was quantified by measuring fresh weight of flg22-treated seedlings as a percentage of water-treated controls (n = 4). Error bars in A, B, C, D, and F, represent se. Lowercase letters represent significantly different statistical groups by Tukey’s honest significant difference test (P < 0.05). Experiments were replicated three times with similar results.Overall, our results support an ETI-specific role for ZRKs in Arabidopsis, acting as sensors of the ZAR1 NLR. Structural insights have revealed important residues required for ZAR1-ZRK1 complex formation, and these are conserved across the RLCK XII-2 family, which includes ZRKs outside the genomic cluster (Supplemental Fig. S6; Lewis et al., 2013; Wang et al., 2019). This suggests that the ZRKs outside this genomic cluster may also play a similar role as ZAR1 sensors. As such, the ZRK family would have evolved to mimic and/or interact with the numerous kinase virulence targets of pathogenic effectors, thereby expanding the surveillance potential of ZAR1.Supplemental DataThe following supplemental materials are available.

• Supplemental Figure S1. Sequencing confirmation of the ZRK gene cluster deletion.
• Supplemental Figure S2. Fresh weight of zrk knockout (KO) plants (zrk_3.10 and zrk_2.11) relative to wild-type Columbia-0 (wild-type Col-0) and zar1-1 plants.
• Supplemental Figure S3. ZRK gene cluster deletion specifically compromises ZRK-dependent ETI responses.
• Supplemental Figure S4. ZRK gene cluster compromises the ZAR1-dependent ETI responses against HopBA1a, HopO1c, and HopX1i.
• Supplemental Figure S5. Bacterial growth of the virulent PtoDC3000 strain on zrk KO plants (zrk_3.10 and zrk_2.11) relative to wild-type Col-0 (wild-type Col-0) plants 0- and 3-d post-inoculation via syringe infiltration.
• Supplemental Figure S6. Multiple sequence alignment of RLCK XII-2 family shows high conservation of putative ZAR1-interacting residues.
• Supplemental Methods S1. Generation and characterization of ZRK cluster deletion lines.

     

The Arabidopsis Vacuolar Sorting Receptor1 Is Required for Osmotic Stress-Induced Abscisic Acid Biosynthesis

Osmotic stress activates the biosynthesis of the phytohormone abscisic acid (ABA) through a pathway that is rate limited by the carotenoid cleavage enzyme 9-cis-epoxycarotenoid dioxygenase (NCED). To understand the signal transduction mechanism underlying the activation of ABA biosynthesis, we performed a forward genetic screen to isolate mutants defective in osmotic stress regulation of the NCED3 gene. Here, we identified the Arabidopsis (Arabidopsis thaliana) Vacuolar Sorting Receptor1 (VSR1) as a unique regulator of ABA biosynthesis. The vsr1 mutant not only shows increased sensitivity to osmotic stress, but also is defective in the feedback regulation of ABA biosynthesis by ABA. Further analysis revealed that vacuolar trafficking mediated by VSR1 is required for osmotic stress-responsive ABA biosynthesis and osmotic stress tolerance. Moreover, under osmotic stress conditions, the membrane potential, calcium flux, and vacuolar pH changes in the vsr1 mutant differ from those in the wild type. Given that manipulation of the intracellular pH is sufficient to modulate the expression of ABA biosynthesis genes, including NCED3, and ABA accumulation, we propose that intracellular pH changes caused by osmotic stress may play a signaling role in regulating ABA biosynthesis and that this regulation is dependent on functional VSR1.Plant vacuoles are vital organelles for maintaining cell volume and cell turgor, regulating ion homeostasis and pH, disposing toxic materials, and storing and degrading unwanted proteins (Marty, 1999). To perform these diverse functions, vacuoles require an array of different and complex proteins. These proteins are synthesized at the endoplasmic reticulum (ER) and are transported to the vacuole through the vacuolar trafficking pathway. Perturbation of the vacuolar trafficking machinery affects many cellular processes, including tropisms, responses to pathogens, cytokinesis, hormone transport, and signal transduction (Surpin and Raikhel, 2004). The vacuolar trafficking system is comprised of several compartments: the ER, the Golgi apparatus, the trans-Golgi network (TGN), the prevacuolar compartment (PVC), and the vacuole. Vacuolar proteins synthesized at the ER are transported to the cis-Golgi via coat protein complex II (COPII) vesicles and are then transported to the TGN through the Golgi apparatus. In the TGN, proteins are sorted for delivery to their respective locations according to their targeting signal. Vacuolar proteins carrying a vacuolar sorting signal are thought to be recognized by vacuolar sorting receptors (VSRs), which are mainly located in the PVC, although sorting of vacuolar proteins may also occur at the ER and VSRs can be recycled from the TGN to the ER (Castelli and Vitale, 2005; Niemes et al., 2010). Multiple studies suggest that plant VSRs serve as sorting receptors both for lytic vacuole proteins (daSilva et al., 2005; Foresti et al., 2006; Kim et al., 2010) and for storage vacuole proteins (Shimada et al., 2003; Fuji et al., 2007; Zouhar et al., 2010).Osmotic stress is commonly associated with many environmental stresses, including drought, cold, and high soil salinity, that have a severe impact on the productivity of agricultural plants worldwide. Therefore, understanding how plants perceive and respond to osmotic stress is critical for improving plant resistance to abiotic stresses (Zhu, 2002; Fujita et al., 2013). It has long been recognized that osmotic stress can activate several signaling pathways that lead to changes in gene expression and metabolism. One important regulator of these signaling pathways is the phytohormone abscisic acid (ABA), which accumulates in response to osmotic stress. ABA regulates many critical processes, such as seed dormancy, stomatal movement, and adaptation to environmental stress (Finkelstein and Gibson, 2002; Xiong and Zhu, 2003; Cutler et al., 2010). De novo synthesis of ABA is of primary importance for increasing ABA levels in response to abiotic stress. ABA is synthesized through the cleavage of a C40 carotenoid originating from the 2-C-methyl-d-erythritol-4-phosphate pathway, followed by a conversion from zeaxanthin to violaxanthin catalyzed by the zeaxanthin epoxidase ABA1 and then to neoxanthin catalyzed by the neoxanthin synthase ABA4. Subsequently, a 9-cis-epoxycarotenoid dioxygenase (NCED) cleaves the violaxanthin and neoxanthin to xanthoxin. Xanthoxin, in turn, is oxidized by a short-chain alcohol dehydrogenase (ABA2) to abscisic aldehyde, which is converted to ABA by abscisic acid aldehyde oxidase3 (AAO3) using a molybdenum cofactor activated by the molybdenum cofactor sulfurase (ABA3; Nambara and Marion-Poll, 2005). In this pathway, it is generally thought that the cleavage step catalyzed by NCED is the rate-limiting step (Iuchi et al., 2000, 2001; Qin and Zeevaart, 2002; Xiong and Zhu, 2003). In Arabidopsis (Arabidopsis thaliana), five members of the NCED family (NCED2, NCED3, NCED5, NCED6, and NCED9) have been characterized (Tan et al., 2003). Of those, NCED3 has been suggested to play a crucial role in ABA biosynthesis, and its expression is induced by dehydration and osmotic stress (Iuchi et al., 2000, 2001; Qin and Zeevaart, 2002; Xiong and Zhu, 2003). Thus, understanding how the NCED3 gene is activated in response to osmotic stress is important for the elucidation of the mechanisms that govern plant acclimation to abiotic stress.We have used the firefly luciferase reporter gene driven by the stress-responsive NCED3 promoter to enable the genetic dissection of plant responses to osmotic stress (Wang et al., 2011). Here, we report the characterization of a unique regulator of ABA biosynthesis, 9-cis Epoxycarotenoid Dioxygenase Defective2 (CED2). The ced2 mutants are impaired in osmotic stress tolerance and are defective in the expression of genes required for ABA synthesis and consequently osmotic stress-induced ABA accumulation. The CED2 gene encodes VSR1, previously known to be involved in vacuolar trafficking but not known to be critical for osmotic stress induction of ABA biosynthesis and osmotic stress tolerance. Our study further suggests that intracellular pH changes might act as an early stress response signal triggering osmotic stress-activated ABA biosynthesis.     

Maternal Control of PIN1 Is Required for Female Gametophyte Development in Arabidopsis

Land plants are characterised by haplo-diploid life cycles, and developing ovules are the organs in which the haploid and diploid generations coexist. Recently it has been shown that hormones such as auxin and cytokinins play important roles in ovule development and patterning. The establishment and regulation of auxin levels in cells is predominantly determined by the activity of the auxin efflux carrier proteins PIN-FORMED (PIN). To study the roles of PIN1 and PIN3 during ovule development we have used mutant alleles of both genes and also perturbed PIN1 and PIN3 expression using micro-RNAs controlled by the ovule specific DEFH9 (DEFIFICENS Homologue 9) promoter. PIN1 down-regulation and pin1-5 mutation severely affect female gametophyte development since embryo sacs arrest at the mono- and/or bi-nuclear stages (FG1 and FG3 stage). PIN3 function is not required for ovule development in wild-type or PIN1-silenced plants. We show that sporophytically expressed PIN1 is required for megagametogenesis, suggesting that sporophytic auxin flux might control the early stages of female gametophyte development, although auxin response is not visible in developing embryo sacs.