Evolution of secondary metabolites, morphological structures and associated gene expression patterns in galls induced by four closely related aphid species on a host plant species.

Gall-forming insects induce various types of galls on their host plants by altering gene expression in host plant organs, and recent studies have been conducted for gene expression in galls. However, the evolutionary trajectories of gene expression patterns and the resulting phenotypes have not yet been studied using multiple related species. We investigated the speciation and the diversification process of galls induced by four closely related aphid species (Hormaphidini) on a host plant species (Hamamelis japonica) by examining the phylogenetic congruence between the geographical divergences of aphids and the host plant, and by comparing their gene expression patterns and resulting phenotypes. Phylogenetic analysis of aphids and the host plant showed that geographical isolation among host plant populations has interrupted gene flow in aphids and accelerated the speciation process. The concentration of phenolics and the complexity of the internal structure of galls were correlated with the expression levels of genes for the biosynthesis of phenolics and morphogenesis respectively. These results suggest that the expression levels of genes for the biosynthesis of phenolics and morphogenesis have evolutionarily increased in galls accelerated by the speciation process of aphids due to the distribution change of the host plant, leading to the related phenotypic evolution. Our study showed the evolutionary process of phenotypic traits in galls in the wild from both gene expression and actual phenotype levels.

VND Genes Redundantly Regulate Cell Wall Thickening during Parasitic Nematode Infection.

Plant parasitic root-knot nematodes are major agricultural pests worldwide, as they infect plant roots and cause substantial damages to crop plants. Root-knot nematodes induce specialized feeding cells known as giant cells (GCs) in the root vasculature, which serve as nutrient reservoirs for the infecting nematodes. Here, we show that the cell walls of GCs thicken to form pitted patterns that superficially resemble metaxylem cells. Interestingly, VASCULAR-RELATED NAC-DOMAIN1 (VND1) was found to be upregulated, while the xylem-type programmed cell death marker XYLEM CYSTEINE PEPTIDASE 1 was downregulated upon nematode infection. The vnd2 and vnd3 mutants showed reduced secondary cell wall pore size, while the vnd1 vnd2 vnd3 triple mutant produced significantly fewer nematode egg masses when compared with the wild type. These results suggest that the GC development pathway likely shares common signaling modules with the metaxylem differentiation pathway and VND1, VND2, and VND3 redundantly regulate plant-nematode interaction through secondary cell wall formation.

Synovial-tissue resident macrophages play proinflammatory functions in the pathogenesis of RA while maintaining the phenotypes in the steady state.

Synovial tissue-resident macrophages (STRMs) maintain normal joint homeostasis in a steady state. However, it is unclear whether STRMs still play homeostatic roles or change the functions in the joint of rheumatoid arthritis (RA), where infiltrating peripheral blood monocyte-derived macrophages (PBMoMs) play proinflammatory roles. In the present study, we examined changes in the phenotypes and functions of STRMs in response to RA-related stimuli in vitro. STRMs were prepared from non-inflammatory osteoarthritis (OA) joint synovium, which is histologically indistinguishable from normal joint synovium. PBMoMs were prepared and used for comparison. After stimulation with plate-bound IgG, which mimics anti-citrullinated protein antibody immunocomplex formed in RA joints, or with combinations of RA-related inflammatory mediators, namely tumor necrosis factor-α (TNF-α) and prostaglandin E2 or interferon-γ, PBMoMs downregulated surface markers and genes associated with anti-inflammatory macrophages, and upregulated cytokine and marker genes of proinflammatory macrophages in RA. On the other hand, STRMs hardly changed the expression of surface molecules and marker genes but altered the pattern of cytokine gene expression after stimulation like PBMoMs. Furthermore, in vitro stimulated STRMs promote proinflammatory functions of cocultured synovial fibroblasts. Thus, STRMs might play proinflammatory roles in RA joints, while maintaining their phenotypes in the steady state.

Dissection of the IDA promoter identifies WRKY transcription factors as abscission regulators in Arabidopsis.

Plants shed organs such as leaves, petals, or fruits through the process of abscission. Monitoring cues such as age, resource availability, and biotic and abiotic stresses allow plants to abscise organs in a timely manner. How these signals are integrated into the molecular pathways that drive abscission is largely unknown. The INFLORESCENCE DEFICIENT IN ABSCISSION (IDA) gene is one of the main drivers of floral organ abscission in Arabidopsis and is known to transcriptionally respond to most abscission-regulating cues. By interrogating the IDA promoter in silico and in vitro, we identified transcription factors that could potentially modulate IDA expression. We probed the importance of ERF- and WRKY-binding sites for IDA expression during floral organ abscission, with WRKYs being of special relevance to mediate IDA up-regulation in response to biotic stress in tissues destined for separation. We further characterized WRKY57 as a positive regulator of IDA and IDA-like gene expression in abscission zones. Our findings highlight the promise of promoter element-targeted approaches to modulate the responsiveness of the IDA signaling pathway to harness controlled abscission timing for improved crop productivity.

Root-knot nematode modulates plant CLE3-CLV1 signaling as a long-distance signal for successful infection.

Plants use many long-distance and systemic signals to modulate growth and development, as well as respond to biotic and abiotic stresses. Parasitic nematodes infect host plant roots and cause severe damage to crop plants. However, the molecular mechanisms that regulate parasitic nematode infections are still unknown. Here, we show that plant parasitic root-knot nematodes (RKNs), Meloidogyne incognita, modulate the host CLAVATA3 (CLV3)/EMBRYO SURROUNDING REGION (CLE)-CLV1 signaling module to promote the infection progression. Plants deficient in the CLE signaling pathway show enhanced RKN resistance, whereas CLE overexpression leads to increased susceptibility toward RKN. Grafting analysis shows that CLV1 expression in the shoot alone is sufficient to positively regulate RKN infection. Together with results from the split-root culture system, infection assays, and CLE3-CLV1 binding assays, we conclude that mobile root-derived CLE signals are perceived by CLV1 in the shoot, which subsequently produce systemic signals to promote gall formation and RKN reproduction.

A mechanical theory of competition between plant root growth and soil pressure reveals a potential mechanism of root penetration.

Root penetration into the soil is essential for plants to access water and nutrients, as well as to mechanically support aboveground structures. This requires a combination of healthy plant growth, adequate soil mechanical properties, and compatible plant-soil interactions. Despite the current knowledge of the static rheology driving the interactions at the root-soil interface, few theoretical approaches have attempted to describe root penetration with dynamic rheology. In this work, we experimentally showed that radish roots in contact with soil of specific density during a specific growth stage fail to penetrate the soil. To explore the mechanism of root penetration into the soil, we constructed a theoretical model to explore the relevant conditions amenable to root entry into the soil. The theory indicates that dimensionless parameters such as root growth anisotropy, static root-soil competition, and dynamic root-soil competition are important for root penetration. The consequent theoretical expectations were supported by finite element analysis, and a potential mechanism of root penetration into the soil is discussed.

NAC103 mutation alleviates DNA damage in an Arabidopsis thaliana mutant sensitive to excess boron.

Excess boron (B) is toxic to plants and thereby causes DNA damage and cell death in root meristems. However, the underlying mechanisms which link boron and DNA damage remain unclear. It has been reported that the rpt5a-6 mutant of the 26S proteasome is sensitive to excess boron, resulting in more frequent cell death in root meristem and reduced root elongation. In this study, we showed that a reduction in root growth in the rpt5a mutant in the presence of high boron levels is repressed by a mutation in NAC domain containing transcription factor NAC103, a substrate of the proteasome, which functions in the unfolded protein response pathway. The mutation in NAC103 alleviated excess-B-induced DNA damage and cell death in root meristems of the rpt5a mutant. Superoxide ( O 2 - ) staining with nitroblue tetrazolium revealed that boron stress causes O 2 - accumulation in root tips, which was higher in the rpt5a-6 mutant, whereas the accumulation was lower in the rpt5a-6 nac103-3 double mutant. Our work demonstrates the overall involvement of NAC103 in maintaining healthy root meristem under excess boron conditions in the absence of RPT5A proteasome subunit.

PECT1, a rate-limiting enzyme in phosphatidylethanolamine biosynthesis, is involved in the regulation of stomatal movement in Arabidopsis.

An Arabidopsis mutant displaying impaired stomatal responses to CO2 , cdi4, was isolated by a leaf thermal imaging screening. The mutated gene PECT1 encodes CTP:phosphorylethanolamine cytidylyltransferase. The cdi4 exhibited a decrease in phosphatidylethanolamine levels and a defect in light-induced stomatal opening as well as low-CO2 -induced stomatal opening. We created RNAi lines in which PECT1 was specifically repressed in guard cells. These lines are impaired in their stomatal responses to low-CO2 concentrations or light. Fungal toxin fusicoccin (FC) promotes stomatal opening by activating plasma membrane H+ -ATPases in guard cells via phosphorylation. Arabidopsis H+ -ATPase1 (AHA1) has been reported to be highly expressed in guard cells, and its activation by FC induces stomatal opening. The cdi4 and PECT1 RNAi lines displayed a reduced stomatal opening response to FC. However, similar to in the wild-type, cdi4 maintained normal levels of phosphorylation and activation of the stomatal H+ -ATPases after FC treatment. Furthermore, the cdi4 displayed normal localization of GFP-AHA1 fusion protein and normal levels of AHA1 transcripts. Based on these results, we discuss how PECT1 could regulate CO2 - and light-induced stomatal movements in guard cells in a manner that is independent and downstream of the activation of H+ -ATPases. [Correction added on 15 May 2023, after first online publication: The third sentence is revised in this version.].

Single-cell RNA sequencing of intestinal immune cells in neonatal necrotizing enterocolitis.

PURPOSE: Necrotizing enterocolitis (NEC) causes fatal intestinal necrosis in neonates, but its etiology is unknown. We analyzed the intestinal immune response to NEC. METHODS: Using single-cell RNA sequencing (scRNA-seq), we analyzed the gene expression profiles of intestinal immune cells from four neonates with intestinal perforation (two with NEC and two without NEC). Target mononuclear cells were extracted from the lamina propria of the resected intestines. RESULTS: In all four cases, major immune cells, such as T cells (15.1-47.7%), B cells (3.1-19.0%), monocytes (16.5-31.2%), macrophages (1.6-17.4%), dendritic cells (2.4-12.2%), and natural killer cells (7.5-12.8%), were present in similar proportions to those in the neonatal cord blood. Gene set enrichment analysis showed that the MTOR, TNF-α, and MYC signaling pathways were enriched in T cells of the NEC patients, suggesting upregulated immune responses related to inflammation and cell proliferation. In addition, all four cases exhibited a bias toward cell-mediated inflammation, based on the predominance of T helper 1 cells. CONCLUSION: Intestinal immunity in NEC subjects exhibited stronger inflammatory responses compared to non-NEC subjects. Further scRNA-seq and cellular analysis may improve our understanding of the pathogenesis of NEC.

Contribution of vasculature to stem integrity in Arabidopsis thaliana.

In plants, coordinated growth is important for organ mechanical integrity because cells remain contiguous through their walls. So far, defects in inflorescence stem integrity in Arabidopsis thaliana have mainly been related to epidermal defects. Although these observations suggest a growth-limiting function at the stem cortex, deeper layers of the stem could also contribute to stem integrity. The nac secondary cell wall thickening promoting factor1 (nst1) nst3 double-mutant background is characterized by weaker vascular bundles without cracks. By screening for the cracking phenotype in this background, we identified a regulator of stem cracking, the transcription factor INDETERMINATE DOMAIN9 (IDD9). Stem cracking was not caused by vascular bundle breakage in plants that expressed a dominant repressor version of IDD9. Instead, cracking emerged from increased cell expansion in non-lignified interfascicular fiber cells that stretched the epidermis. This phenotype could be enhanced through CLAVATA3-dependent cell proliferation. Collectively, our results demonstrate that stem integrity relies on three additive mechanical components: the epidermis, which resists inner cell growth; cell proliferation in inner tissues; and growth heterogeneity associated with vascular bundle distribution in deep tissues.

CLE3 and its homologs share overlapping functions in the modulation of lateral root formation through CLV1 and BAM1 in Arabidopsis thaliana.

Lateral roots are important for a wide range of processes, including uptake of water and nutrients. The CLAVATA3 (CLV3)/EMBRYO SURROUNDING REGION-RELATED (CLE) 1 ~ 7 peptide family and their cognate receptor CLV1 have been shown to negatively regulate lateral root formation under low-nitrate conditions. However, little is known about how CLE signaling regulates lateral root formation. A persistent obstacle in CLE peptide research is their functional redundancies, which makes functional analyses difficult. To address this problem, we generate the cle1 ~ 7 septuple mutant (cle1 ~ 7-cr1, cr stands for mutant allele generated with CRISPR/Cas9). cle1 ~ 7-cr1 exhibits longer lateral roots under normal conditions. Specifically, in cle1 ~ 7-cr1, the lateral root density is increased, and lateral root primordia initiation is found to be accelerated. Further analysis shows that cle3 single mutant exhibits slightly longer lateral roots. On the other hand, plants that overexpress CLE2 and CLE3 exhibit decreased lateral root lengths. To explore cognate receptor(s) of CLE2 and CLE3, we analyze lateral root lengths in clv1 barely any meristem 1(bam1) double mutant. Mutating both the CLV1 and BAM1 causes longer lateral roots, but not in each single mutant. In addition, genetic analysis reveals that CLV1 and BAM1 are epistatic to CLE2 and CLE3. Furthermore, gene expression analysis shows that the LATERAL ORGAN BOUNDARIES DOMAIN/ASYMMETRIC LEAVES2-LIKE (LBD/ASL) genes, which promote lateral root formation, are upregulated in cle1 ~ 7-cr1 and clv1 bam1. We therefore propose that CLE2 and CLE3 peptides are perceived by CLV1 and BAM1 to mediate lateral root formation through LBDs regulation.

A rapid method for detection of the root-knot nematode resistance gene, Mi-1.2, in tomato cultivars.

Molecular markers have been widely used in plant breeding to improve the accuracy and efficiency of trait selection. In particular, molecular markers are powerful in facilitating the introgression of resistance genes by circumventing costly and time-consuming infection assays. To achieve their practical use, it is important to ensure the tight linkage between the markers and the traits. Here we report a new cleaved amplified polymorphic sequence (CAPS) marker, Mi1713, for the root-knot nematode resistance gene, Mi-1.2, in cultivated tomato. The Mi1713 marker is designed in the conserved region of Mi-1.2 and its homologs in tomato and other nightshade species. Combined with a single-step procedure for preparing PCR templates, the Mi1713 marker enables rapid and reliable screening for the presence of Mi-1.2. The approach described in this study is applicable in designing CAPS markers for various genes or alleles of interest in tomato and other crops.

Local auxin synthesis mediated by YUCCA4 induced during root-knot nematode infection positively regulates gall growth and nematode development.

Parasites and pathogens are known to manipulate the host's endogenous signaling pathways to facilitate the infection process. In particular, plant-parasitic root-knot nematodes (RKN) are known to elicit auxin response at the infection sites, to aid the development of root galls as feeding sites for the parasites. Here we describe the role of local auxin synthesis induced during RKN infection. Exogenous application of auxin synthesis inhibitors decreased RKN gall formation rates, gall size and auxin response in galls, while auxin and auxin analogues produced the opposite effects, re-enforcing the notion that auxin positively regulates RKN gall formation. Among the auxin biosynthesis enzymes, YUCCA4 (YUC4) was found to be dramatically up-regulated during RKN infection, suggesting it may be a major contributor to the auxin accumulation during gall formation. However, yuc4-1 showed only very transient decrease in gall auxin levels and did not show significant changes in RKN infection rates, implying the loss of YUC4 is likely compensated by other auxin sources. Nevertheless, yuc4-1 plants produced significantly smaller galls with fewer mature females and egg masses, confirming that auxin synthesized by YUC4 is required for proper gall formation and RKN development within. Interestingly, YUC4 promoter was also activated during cyst nematode infection. These lines of evidence imply auxin biosynthesis from multiple sources, one of them being YUC4, is induced upon plant endoparasitic nematode invasion and likely contribute to their infections. The coordination of these different auxins adds another layer of complexity of hormonal regulations during plant parasitic nematode interaction.

A group of CLE peptides regulates de novo shoot regeneration in Arabidopsis thaliana.

Known for their regulatory roles in stem cell homeostasis, CLAVATA3/ESR-RELATED (CLE) peptides also function as mediators of external stimuli such as hormones. De novo shoot regeneration, representing the remarkable plant cellular plasticity, involves reconstitution of stem cells under control of stem-cell regulators. Yet whether and how stem cell-regulating CLE peptides are implicated in plant regeneration remains unknown. By CRISPR/Cas9-induced loss-of-function studies, peptide application, precursor overexpression, and expression analyses, the role of CLE1-CLE7 peptides and their receptors in de novo shoot regeneration was studied in Arabidopsis thaliana. CLE1-CLE7 are induced by callus-induction medium and dynamically expressed in pluripotent callus. Exogenously-applied CLE1-CLE7 peptides or precursor overexpression effectively leads to shoot regeneration suppression, whereas their simultaneous mutation results in enhanced regenerative capacity, demonstrating that CLE1-CLE7 peptides redundantly function as negative regulators of de novo shoot regeneration. CLE1-CLE7-mediated shoot regeneration suppression is impaired in loss-of-function mutants of callus-expressed CLAVATA1 (CLV1) and BARELY ANY MERISTEM1 (BAM1) genes, indicating that CLV1/BAM1 are required for CLE1-CLE7-mediated shoot regeneration signaling. CLE1-CLE7 signaling resulted in transcriptional repression of WUSCHEL (WUS), a stem cell-promoting transcription factor known as a principal regulator of plant regeneration. Our results indicate that functionally-redundant CLE1-CLE7 peptides genetically act through CLV1/BAM1 receptors and repress WUS expression to modulate shoot-regeneration capacity, establishing the mechanistic basis for CLE1-CLE7-mediated shoot regeneration and a novel role for CLE peptides in hormone-dependent developmental plasticity.

Control of Root Stem Cell Differentiation and Lateral Root Emergence by CLE16/17 Peptides in Arabidopsis.

Secreted peptide-mediated cell-to-cell communication plays a crucial role in the development of multicellular organisms. A large number of secreted peptides have been predicated by bioinformatic approaches in plants. However, only a few of them have been functionally characterized. In this study, we show that two CLAVATA3/EMBRYO SURROUNDING REGION-RELATED (CLE) peptides CLE16/17 are required for both stem cell differentiation and lateral root (LR) emergence in Arabidopsis. We further demonstrate that the CLE16/17 peptides act through the CLAVATA1-ARABIDOPSIS CRINKLY4 (CLV1-ACR4) protein kinase complex in columella stem cell (CSC) differentiation, but not in LR emergence. Furthermore, we show that CLE16/17 promote LR emergence probably via activating the expression of HAESA/HAESA-LIKE2 (HAE/HSL2) required for cell wall remodeling. Collectively, our results reveal a CLV1-ACR4-dependent and -independent dual-function of the CLE16/17 peptides in root development.

Long-distance translocation of CLAVATA3/ESR-related 2 peptide and its positive effect on roots sucrose status.

In vascular plants, roots anchor themselves into the soil and take up water and nutrients to provide them to the shoots. Therefore, continuous growth and development of the roots are important for plant life. To achieve this, photosynthesizing leaves must be able to supply sufficient photoassimilates to the roots. However, the mechanisms by which plants maintain carbon levels in roots remain elusive. Here, we focused on the Arabidopsis (Arabidopsis thaliana) CLAVATA3/ESR-related 2 (CLE2) peptide, which was detected in Arabidopsis xylem exudate, and its homologs. CLE2 and CLE3 genes responded to carbon-deficient conditions. Loss- and gain-of-function mutant analyses showed that CLE genes positively affected root sucrose level. Mutations in the CLE genes resulted in a high shoot/root ratio under sucrose-free conditions. Grafting experiments demonstrated the systemic effect of CLE peptide genes. These findings provide insights into the molecular basis for the relationship between roots and leaves in maintenance of the root sucrose levels and growth.

CLE42 delays leaf senescence by antagonizing ethylene pathway in Arabidopsis.

Leaf senescence is the final stage of leaf development and is influenced by numerous internal and environmental factors. CLE family peptides are plant-specific peptide hormones that regulate various developmental processes. However, the role of CLE in regulating Arabidopsis leaf senescence remains unclear. Here, we found that CLE42 is a negative regulator of leaf senescence by using a CRISPR/Cas9-produced CLE mutant collection. The cle42 mutant displayed earlier senescence phenotypes, while overexpression of CLE42 delayed age-dependent and dark-induced leaf senescence. Moreover, application of the synthesized 12-amino-acid peptide (CLE42p) also delayed leaf senescence under natural and dark conditions. CLE42 and CLE41/44 displayed functional redundancy in leaf senescence, and the cle41 cle42 cle44 triple mutant displayed more pronounced earlier senescence phenotypes than any single mutant. Analysis of differentially expressed genes obtained by RNA-Seq methodology revealed that the ethylene pathway was suppressed by overexpressing CLE42. Moreover, CLE42 suppressed ethylene biosynthesis and thus promoted the protein accumulation of EBF, which in turn decreased the function of EIN3. Accordingly, mutation of EIN3/EIL1 or overexpression of EBF1 suppressed the earlier senescence phenotypes of the cle42 mutant. Together, our results reveal that the CLE peptide hormone regulates leaf senescence by communicating with the ethylene pathway.

Control of Fusarium and nematodes by entomopathogenic fungi for organic production of Zingiber officinale.

Ginger (genus Zingiber) is widely used as a spice and a medicinal herb worldwide and is the major ingredient of traditional local drinks such as jamu in Southeast Asia. Because ginger is frequently consumed, there is an increasing interest in organic ginger production without the use of synthetic agrochemicals. Recent studies have reported that certain kinds of entomopathogenic fungi (EPF) can establish endophytic- or mycorrhiza-like relationships with plants, thereby promoting plant growth and health, in addition to their typical role in crop protection as biological control agents. In this study, we explored the possibility of non-entomopathogenic effects of EPF Beauveria bassiana and Cordyceps fumosorosea on ginger plants (Zingiber officinale) via antagonism with Fusarium oxysporum or the parasitic nematode Meloidogyne incognita. The two EPF negatively affected the growth of F. oxysporum and survival of M. incognita in vitro. The application of EPF did not have any negative effect on the growth of ginger plants. Soil chemical properties were not different between the plots with or without EPF application, while the diversity of soil bacteria was observed to increase on application of EPF. At least C. fumosorosea appeared to persist in soil during the period of ginger cultivation. Thus, these EPF are potentially useful tools for producing chemical-free ginger.

Rhamnogalacturonan-I as a nematode chemoattractant from Lotus corniculatus L. super-growing root culture.

Introduction: The soil houses a tremendous amount of micro-organisms, many of which are plant parasites and pathogens by feeding off plant roots for sustenance. Such root pathogens and parasites often rely on plant-secreted signaling molecules in the rhizosphere as host guidance cues. Here we describe the isolation and characterization of a chemoattractant of plant-parasitic root-knot nematodes (Meloidogyne incognita, RKN). Methods: The Super-growing Root (SR) culture, consisting of excised roots from the legume species Lotus corniculatus L., was found to strongly attract infective RKN juveniles and actively secrete chemoattractants into the liquid culture media. The chemo-attractant in the culture media supernatant was purified using hydrophobicity and anion exchange chromatography, and found to be enriched in carbohydrates. Results: Monosaccharide analyses suggest the chemo-attractant contains a wide array of sugars, but is enriched in arabinose, galactose and galacturonic acid. This purified chemoattractant was shown to contain pectin, specifically anti-rhamnogalacturonan-I and anti-arabinogalactan protein epitopes but not anti-homogalacturonan epitopes. More importantly, the arabinose and galactose sidechain groups were found to be essential for RKN-attracting activities. This chemo-attractant appears to be specific to M. incognita, as it wasn't effective in attracting other Meloidogyne species nor Caenorhabditis elegans. Discussion: This is the first report to identify the nematode attractant purified from root exudate of L corniculatus L. Our findings re-enforce pectic carbohydrates as important chemicals mediating micro-organism chemotaxis in the soil, and also highlight the unexpected utilities of the SR culture system in root pathogen research.