BAK1 protects the receptor-like kinase BIR2 from SNIPER2a/b-mediated degradation to promote pattern-triggered immunity in Nicotiana benthamiana.

The detection of microbial infections by plants induces the rapid formation of immune receptor complexes at the plasma membrane. However, how this process is controlled to ensure proper immune signaling remains largely unknown. Here, we found that the Nicotiana benthamiana membrane-localized leucine-rich repeat receptor-like kinase BAK1-INTERACTING RLK 2 (NbBIR2) constitutively associates with BRI1-ASSOCIATED RECEPTOR KINASE 1 (NbBAK1) in vivo and in vitro and promotes complex formation with pattern recognition receptors. In addition, NbBIR2 is targeted by 2 RING-type ubiquitin E3 ligases, SNC1-INFLUENCING PLANT E3 LIGASE REVERSE 2a (NbSNIPER2a) and NbSNIPER2b, for ubiquitination and subsequent degradation in planta. NbSNIPER2a and NbSNIPER2b interact with NbBIR2 in vivo and in vitro and are released from NbBIR2 upon treatment with different microbial patterns. Furthermore, accumulation of NbBIR2 in response to microbial patterns is tightly associated with NbBAK1 abundance in N. benthamiana. NbBAK1 acts as a modular protein that stabilizes NbBIR2 by competing with NbSNIPER2a or NbSNIPER2b for association with NbBIR2. Similar to NbBAK1, NbBIR2 positively regulates pattern-triggered immunity and resistance to bacterial and oomycete pathogens in N. benthamiana, whereas NbSNIPER2a and NbSNIPER2b have the opposite effect. Together, these results reveal a feedback regulatory mechanism employed by plants to tailor pattern-triggered immune signaling.

The Phytophthora sojae nuclear effector PsAvh110 targets a host transcriptional complex to modulate plant immunity.

Plants have evolved sophisticated immune networks to restrict pathogen colonization. In response, pathogens deploy numerous virulent effectors to circumvent plant immune responses. However, the molecular mechanisms by which pathogen-derived effectors suppress plant defenses remain elusive. Here, we report that the nucleus-localized RxLR effector PsAvh110 from the pathogen Phytophthora sojae, causing soybean (Glycine max) stem and root rot, modulates the activity of a transcriptional complex to suppress plant immunity. Soybean like-heterochromatin protein 1-2 (GmLHP1-2) and plant homeodomain finger protein 6 (GmPHD6) form a transcriptional complex with transcriptional activity that positively regulates plant immunity against Phytophthora infection. To suppress plant immunity, the nuclear effector PsAvh110 disrupts the assembly of the GmLHP1-2/GmPHD6 complex via specifically binding to GmLHP1-2, thus blocking its transcriptional activity. We further show that PsAvh110 represses the expression of a subset of immune-associated genes, including BRI1-associated receptor kinase 1-3 (GmBAK1-3) and pathogenesis-related protein 1 (GmPR1), via G-rich elements in gene promoters. Importantly, PsAvh110 is a conserved effector in different Phytophthora species, suggesting that the PsAvh110 regulatory mechanism might be widely utilized in the genus to manipulate plant immunity. Thus, our study reveals a regulatory mechanism by which pathogen effectors target a transcriptional complex to reprogram transcription.

An atypical Phytophthora sojae RxLR effector manipulates host vesicle trafficking to promote infection.

In plants, the apoplast is a critical battlefield for plant-microbe interactions. Plants secrete defense-related proteins into the apoplast to ward off the invasion of pathogens. How microbial pathogens overcome plant apoplastic immunity remains largely unknown. In this study, we reported that an atypical RxLR effector PsAvh181 secreted by Phytophthora sojae, inhibits the secretion of plant defense-related apoplastic proteins. PsAvh181 localizes to plant plasma membrane and essential for P. sojae infection. By co-immunoprecipitation assay followed by liquid chromatography-tandem mass spectrometry analyses, we identified the soybean GmSNAP-1 as a candidate host target of PsAvh181. GmSNAP-1 encodes a soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein, which associates with GmNSF of the SNARE complex functioning in vesicle trafficking. PsAvh181 binds to GmSNAP-1 in vivo and in vitro. PsAvh181 interferes with the interaction between GmSNAP-1 and GmNSF, and blocks the secretion of apoplastic defense-related proteins, such as pathogenesis-related protein PR-1 and apoplastic proteases. Taken together, these data show that an atypical P. sojae RxLR effector suppresses host apoplastic immunity by manipulating the host SNARE complex to interfere with host vesicle trafficking pathway.

The GDSL Lipase MHZ11 Modulates Ethylene Signaling in Rice Roots.

Ethylene plays important roles in plant growth and development, but the regulation of ethylene signaling is largely unclear, especially in crops such as rice (Oryza sativa). Here, by analysis of the ethylene-insensitive mutant mao huzi 11 (mhz11), we identified the GDSL lipase MHZ11, which modulates ethylene signaling in rice roots. MHZ11 localized to the endoplasmic reticulum membrane and has acyl-hydrolyzing activity. This activity affects the homeostasis of sterols in rice roots and is required for root ethylene response. MHZ11 overexpression caused constitutive ethylene response in roots. Genetically, MHZ11 acts with the ethylene receptor ETHYLENE RESPONSE SENSOR2 (OsERS2) upstream of CONSTITUTIVE TRIPLE RESPONSE2 (OsCTR2) and ETHYLENE INSENSITIVE2 (OsEIN2). The mhz11 mutant maintains more OsCTR2 in the phosphorylated form whereas MHZ11 overexpression promotes ethylene-mediated inhibition of OsCTR2 phosphorylation. MHZ11 colocalized with the ethylene receptor OsERS2, and its effect on OsCTR2 phosphorylation requires ethylene perception and initiation of ethylene signaling. The mhz11 mutant overaccumulated sterols and blocking sterol biosynthesis partially rescued the mhz11 ethylene response, likely by reducing receptor-OsCTR2 interaction and OsCTR2 phosphorylation. We propose that MHZ11 reduces sterol levels to impair receptor-OsCTR2 interactions and OsCTR2 phosphorylation for triggering ethylene signaling. Our study reveals a mechanism by which MHZ11 participates in ethylene signaling for regulation of root growth in rice.

CRISPR/Cas9-mediated editing of GmTAP1 confers enhanced resistance to Phytophthora sojae in soybean.

Soybean root rot disease caused by Phytophthora sojae seriously constrains soybean yield. Knocking out the susceptibility gene GmTAP1 in soybean created new soybean lines resistant to several P. sojae strains and these lines showed no agronomic penalties in the field.

Large chromosomal segment deletions by CRISPR/LbCpf1-mediated multiplex gene editing in soybean.

The creation of new soybean varieties has been limited by genomic duplication and redundancy. Efficient multiplex gene editing and large chromosomal segment deletion through clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) systems are promising strategies for overcoming these obstacles. CRISPR/Cpf1 is a robust tool for multiplex gene editing. However, large chromosomal excision mediated by CRISPR/Cpf1 has been reported in only a few non-plant species. Here, we report on CRISPR/LbCpf1-induced large chromosomal segment deletions in soybean using multiplex gene targeting. The CRISPR/LbCpf1 system was optimized for direct repeat and guide RNA lengths in crispr RNA (crRNA) array. The editing efficiency was evaluated using LbCpf1 driven by the CaMV35S and soybean ubiquitin promoter. The optimized system exhibited editing efficiencies of up to 91.7%. Our results showed eight gene targets could be edited simultaneously in one step when a single eight-gRNA-target crRNA array was employed, with an efficiency of up to 17.1%. We successfully employed CRISPR/LbCpf1 to produce small fragments (<1 Kb) and large chromosomal segment deletions (10 Kb-1 Mb) involving four different gene clusters in soybean. Together, these data demonstrate the power of the CRISPR/LbCpf1 platform for multiplex gene editing and chromosomal segment deletion in soybean, supporting the use of this technology in both basic research and agricultural applications.

Transcriptome Profiling of Plant Genes in Response to Agrobacterium tumefaciens-Mediated Transformation.

Agrobacterium tumefaciens is a plant pathogen that causes crown gall disease. During infection of the host plant, Agrobacterium transfers T-DNA from its Ti plasmid into the host cell, which can then be integrated into the host genome. This unique genetic transformation capability has been employed as the dominant technology for producing genetically modified plants for both basic research and biotechnological applications. Agrobacterium has been well studied as a disease-causing agent. The Agrobacterium-mediated transformation process involves early attachment of the bacterium to the host's surface, followed by transfer of T-DNA and virulence proteins into the plant cell. Throughout this process, the host plants exhibit dynamic gene expression patterns at each infection stage or in response to Agrobacterium strains with varying pathogenic capabilities. Shifting host gene expression patterns throughout the transformation process have effects on transformation frequency, host morphology, and metabolism. Thus, gene expression profiling during the Agrobacterium infection process can be an important approach to help elucidate the interaction between Agrobacterium and plants. This review highlights recent findings on host plant differential gene expression patterns in response to A. tumefaciens or related elicitor molecules.

MhMAPK4 from Malus hupehensis Rehd. decreases cell death in tobacco roots by controlling Cd2+ uptake.

Cadmium (Cd) induces cell death in plant roots. Mitogen-activated protein kinase (MAPK) plays a role in the regulation of cell death induced by Cd in plant roots. In this study, MhMAPK4 was isolated from the roots of Malus hupehensis. Subcellular localization showed that the MhMAPK4 protein was located in the cell membrane and cytoplasm and is a transmembrane protein that is characterized by hydrophily. The expression of MhMAPK4 in the roots of M. hupehensis was up-regulated by Cd sulfate and Cd chloride. Phenotypic comparison under Cd stress showed that the growth of wild-type (WT) tobacco was lower than the transgenic lines overexpressing MhMAPK4. The fresh weight and the root length of WT also was lower than that of the transgenic tobacco. The net Cd2+ influx in the tobacco roots was decreased by the overexpression of MhMAPK4, as was root Cd accumulation. The recovery time of the Cd2+ influx to stable state in the transgenic tobacco was also shorter than the WT. The expression of iron-regulated transporter 1 (NtIRT1) and natural resistance associated macrophage protein 5 (NtNRAMP5) was relatively low in the transgenic lines under Cd stress. Cell death and apoptosis in the tobacco roots was reduced following the overexpression of MhMAPK4. The activity of vacuolar processing enzyme (VPE) and the transcript level of VPE in the transgenic tobacco was lower than that of WT under Cd stress. In addition, the electrolyte leakage and malondialdehyde and hydrogen peroxide contents in the transgenic tobacco were lower than those of WT, whereas the antioxidant enzyme activity and expression were higher. These results suggest that MhMAPK4 regulates Cd accumulation by mediating Cd2+ uptake by the roots, and controls Cd-caused cell death by adjusting VPE activity.

Transcriptomic Analysis of Arabidopsis Seedlings in Response to an Agrobacterium-Mediated Transformation Process.

Agrobacterium tumefaciens is a plant pathogen that causes crown gall disease. This pathogen is capable of transferring the T-DNA from its Ti plasmid to the host cell and, then, integrating it into the host genome. To date, this genetic transformation ability has been harnessed as the dominant technology to produce genetically modified plants for both basic research and crop biotechnological applications. However, little is known about the interaction between Agrobacterium tumefaciens and host plants, especially the host responses to Agrobacterium infection and its associated factors. We employed RNA-seq to follow the time course of gene expression in Arabidopsis seedlings infected with either an avirulent or a virulent Agrobacterium strain. Gene Ontology analysis indicated many biological processes were involved in the Agrobacterium-mediated transformation process, including hormone signaling, defense response, cellular biosynthesis, and nucleic acid metabolism. RNAseq and quantitative reverse transcription-polymerase chain reaction results indicated that expression of genes involved in host plant growth and development were repressed but those involved in defense response were induced by Agrobacterium tumefaciens. Further analysis of the responses of transgenic Arabidopsis lines constitutively expressing either the VirE2 or VirE3 protein suggested Vir proteins act to enhance plant defense responses in addition to their known roles facilitating T-DNA transformation.

Ethylene responses in rice roots and coleoptiles are differentially regulated by a carotenoid isomerase-mediated abscisic acid pathway.

Ethylene and abscisic acid (ABA) act synergistically or antagonistically to regulate plant growth and development. ABA is derived from the carotenoid biosynthesis pathway. Here, we analyzed the interplay among ethylene, carotenoid biogenesis, and ABA in rice (Oryza sativa) using the rice ethylene response mutant mhz5, which displays a reduced ethylene response in roots but an enhanced ethylene response in coleoptiles. We found that MHZ5 encodes a carotenoid isomerase and that the mutation in mhz5 blocks carotenoid biosynthesis, reduces ABA accumulation, and promotes ethylene production in etiolated seedlings. ABA can largely rescue the ethylene response of the mhz5 mutant. Ethylene induces MHZ5 expression, the production of neoxanthin, an ABA biosynthesis precursor, and ABA accumulation in roots. MHZ5 overexpression results in enhanced ethylene sensitivity in roots and reduced ethylene sensitivity in coleoptiles. Mutation or overexpression of MHZ5 also alters the expression of ethylene-responsive genes. Genetic studies revealed that the MHZ5-mediated ABA pathway acts downstream of ethylene signaling to inhibit root growth. The MHZ5-mediated ABA pathway likely acts upstream but negatively regulates ethylene signaling to control coleoptile growth. Our study reveals novel interactions among ethylene, carotenogenesis, and ABA and provides insight into improvements in agronomic traits and adaptive growth through the manipulation of these pathways in rice.

MAOHUZI6/ETHYLENE INSENSITIVE3-LIKE1 and ETHYLENE INSENSITIVE3-LIKE2 Regulate Ethylene Response of Roots and Coleoptiles and Negatively Affect Salt Tolerance in Rice.

Ethylene plays important roles in plant growth, development, and stress responses. The ethylene signaling pathway has been studied extensively, mainly in Arabidopsis (Arabidopsis thaliana). However, the molecular mechanism of ethylene signaling is largely unknown in rice (Oryza sativa). Previously, we have isolated a set of rice ethylene-response mutants. Here, we characterized the mutant maohuzi6 (mhz6). Through map-based cloning, we found that MHZ6 encodes ETHYLENE INSENSITIVE3-LIKE1 (OsEIL1), a rice homolog of ETHYLENE INSENSITIVE3 (EIN3), which is the master transcriptional regulator of ethylene signaling in Arabidopsis. Disruption of MHZ6/OsEIL1 caused ethylene insensitivity mainly in roots, whereas silencing of the closely related OsEIL2 led to ethylene insensitivity mainly in coleoptiles of etiolated seedlings. This organ-specific functional divergence is different from the functional features of EIN3 and EIL1, both of which mediate the incomplete ethylene responses of Arabidopsis etiolated seedlings. In Arabidopsis, EIN3 and EIL1 play positive roles in plant salt tolerance. In rice, however, lack of MHZ6/OsEIL1 or OsEIL2 functions improves salt tolerance, whereas the overexpressing lines exhibit salt hypersensitivity at the seedling stage, indicating that MHZ6/OsEIL1 and OsEIL2 negatively regulate salt tolerance in rice. Furthermore, this negative regulation by MHZ6/OsEIL1 and OsEIL2 in salt tolerance is likely attributable in part to the direct regulation of HIGH-AFFINITY K(+) TRANSPORTER2;1 expression and Na(+) uptake in roots. Additionally, MHZ6/OsEIL1 overexpression promotes grain size and thousand-grain weight. Together, our study provides insights for the functional diversification of MHZ6/OsEIL1 and OsEIL2 in ethylene response and finds a novel mode of ethylene-regulated salt stress response that could be helpful for engineering salt-tolerant crops.

An S-domain receptor-like kinase, OsSIK2, confers abiotic stress tolerance and delays dark-induced leaf senescence in rice.

Receptor-like kinases play important roles in plant development and defense responses; however, their functions in other processes remain unclear. Here, we report that OsSIK2, an S-domain receptor-like kinase from rice (Oryza sativa), is involved in abiotic stress and the senescence process. OsSIK2 is a plasma membrane-localized protein with kinase activity in the presence of Mn(2+). OsSIK2 is expressed mainly in rice leaf and sheath and can be induced by NaCl, drought, cold, dark, and abscisic acid treatment. Transgenic plants overexpressing OsSIK2 and mutant sik2 exhibit enhanced and reduced tolerance to salt and drought stress, respectively, compared with the controls. Interestingly, a truncated version of OsSIK2 without most of the extracellular region confers higher salt tolerance than the full-length OsSIK2, likely through the activation of different sets of downstream genes. Moreover, seedlings of OsSIK2-overexpressing transgenic plants exhibit early leaf development and a delayed dark-induced senescence phenotype, while mutant sik2 shows the opposite phenotype. The downstream PR-related genes specifically up-regulated by full-length OsSIK2 or the DREB-like genes solely enhanced by truncated OsSIK2 are all induced by salt, drought, and dark treatments. These results indicate that OsSIK2 may integrate stress signals into a developmental program for better adaptive growth under unfavorable conditions. Manipulation of OsSIK2 should facilitate the improvement of production in rice and other crops.

Multiplex CRISPR/Cas9-mediated raffinose synthase gene editing reduces raffinose family oligosaccharides in soybean.

Soybean [Glycine max (L.) Merr.] is an important world economic crop. It is rich in oil, protein, and starch, and soluble carbohydrates in soybean seeds are also important for human and livestock consumption. The predominant soluble carbohydrate in soybean seed is composed of sucrose and raffinose family oligosaccharides (RFOs). Among these carbohydrates, only sucrose can be digested by humans and monogastric animals and is beneficial for metabolizable energy, while RFOs are anti-nutritional factors in diets, usually leading to flatulence and indigestion, ultimately reducing energy efficiency. Hence, breeding efforts to remove RFOs from soybean seeds can increase metabolizable energy and improve nutritional quality. The objective of this research is to use the multiplex Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9-mediated gene editing system to induce the knockout of soybean raffinose synthase (RS) genes RS2 and RS3 simultaneously to reduce RFOs in mature seeds. First, we constructed five types of multiplex gene editing systems and compared their editing efficiency in soybean hairy roots. We confirmed that the two-component transcriptional unit (TCTU) and single transcriptional unit (STU) systems with transfer RNA (tRNA) as the cleavage site performed better than other systems. The average editing efficiency at the four targets with TCTU-tRNA and STU-tRNA was 50.5% and 46.7%, respectively. Then, we designed four single-guide RNA (sgRNA) targets to induce mutations at RS2 and RS3 by using the TCTU-tRNA system. After the soybean transformation, we obtained several RS2 and RS3 mutation plants, and a subset of alleles was successfully transferred to the progeny. We identified null single and double mutants at the T2 generation and analyzed the seed carbohydrate content of their progeny. The RS2 and RS3 double mutants and the RS2 single mutant exhibited dramatically reduced levels of raffinose and stachyose in mature seeds. Further analysis of the growth and development of these mutants showed that there were no penalties on these phenotypes. Our results indicate that knocking out RS genes by multiplex CRISPR/Cas9-mediated gene editing is an efficient way to reduce RFOs in soybean. This research demonstrates the potential of using elite soybean cultivars to improve the soybean meal trait by multiplex CRISPR(Clustered Regularly Interspaced Short Palindromic Repeats)/Cas9-mediated gene editing.

A Rhodol-based Fluorescent Probe with a Pair of Hydrophilic and Rotatable Wings for Sensitively Monitoring Intracellular Polarity.

Cell polarity, as a vital intracellular microenvironment characteristic, has immense effects on numerous pathological and biological processes. Therefore, the tracking of polarity variations is highly essential to explore the role and mechanism of the polarity in pathophysiological processes. Herein, we designed and synthesized a novel rhodol-based fluorescent probe (RDS) sensitive to polarity by introducing a bis(2-hydroxyethylthio)methyl group, like a pair of hydrophilic and rotatable wings, into the rhodol skeleton. This unique design makes RDS adopt the colorless and non-fluorescent spirocyclic form in a low-polarity medium while the colored and fluorescent ring-open form is present in a high-polarity system, resulting in a positive-correlation response of fluorescence intension to polarity. Importantly, RDS was successfully applied to monitor the polarity changes in living cells including cancer cells, healthy cells and senescent healthy cells, visualizing that the polarity of cancer cells is lower than that of healthy cells in which the more senescent ones have higher polarity.

EIN2 regulates salt stress response and interacts with a MA3 domain-containing protein ECIP1 in Arabidopsis.

Ethylene signalling regulates plant growth and development. However, its roles in salt stress response are less known. Here we studied functions of EIN2, a central membrane protein of ethylene signalling, and its interacting protein ECIP1 in salt stress responses. Mutation of EIN2 led to extreme salt sensitivity as revealed by phenotypic and physiological changes, and overexpression of C-terminus of EIN2 suppressed salt sensitivity in ein2-5, indicating that EIN2 is required for salt tolerance. Downstream components EIN3 and EIL1 are also essential for salt tolerance because ein3-1eil1-1 double mutant showed extreme salt-sensitive phenotype. A MA3 domain-containing protein ECIP1 was further identified to interact with EIN2 in yeast two-hybrid assay and GST pull-down assay. Loss-of-function of ECIP1 resulted in enhanced ethylene response but altered salt response during seed germination and plant growth. Double mutant analysis revealed that ein2-1 was epistatic to ecip1, and ecip1 mutation partially suppressed ethylene-insensitivity of etr2-1 and ein4-1. These studies strengthen that interactions between ECIP1 and EIN2 or ethylene receptors regulate ethylene response and stress response.

MKK4/5-MPK3/6 Cascade Regulates Agrobacterium-Mediated Transformation by Modulating Plant Immunity in Arabidopsis.

Agrobacterium tumefaciens is a specialized plant pathogen that causes crown gall disease and is commonly used for Agrobacterium-mediated transformation. As a pathogen, Agrobacterium triggers plant immunity, which affects transformation. However, the signaling components and pathways in plant immunity to Agrobacterium remain elusive. We demonstrate that two Arabidopsis mitogen-activated protein kinase kinases (MAPKKs) MKK4/MKK5 and their downstream mitogen-activated protein kinases (MAPKs) MPK3/MPK6 play major roles in both Agrobacterium-triggered immunity and Agrobacterium-mediated transformation. Agrobacteria induce MPK3/MPK6 activity and the expression of plant defense response genes at a very early stage. This process is dependent on the MKK4/MKK5 function. The loss of the function of MKK4 and MKK5 or their downstream MPK3 and MPK6 abolishes plant immunity to agrobacteria and increases transformation frequency, whereas the activation of MKK4 and MKK5 enhances plant immunity and represses transformation. Global transcriptome analysis indicates that agrobacteria induce various plant defense pathways, including reactive oxygen species (ROS) production, ethylene (ET), and salicylic acid- (SA-) mediated defense responses, and that MKK4/MKK5 is essential for the induction of these pathways. The activation of MKK4 and MKK5 promotes ROS production and cell death during agrobacteria infection. Based on these results, we propose that the MKK4/5-MPK3/6 cascade is an essential signaling pathway regulating Agrobacterium-mediated transformation through the modulation of Agrobacterium-triggered plant immunity.

Highly efficient Agrobacterium rhizogenes-mediated hairy root transformation for gene functional and gene editing analysis in soybean.

BACKGROUND: Agrobacterium-mediated genetic transformation is a widely used and efficient technique for gene functional research in crop breeding and plant biology. While in some plant species, including soybean, genetic transformation is still recalcitrant and time-consuming, hampering the high-throughput functional analysis of soybean genes. Thus we pursue to develop a rapid, simple, and highly efficient hairy root system induced by Agrobacterium rhizogenes (A. rhizogenes) to analyze soybean gene function. RESULTS: In this report, a rapid, simple, and highly efficient hairy root transformation system for soybean was described. Only sixteen days were required for the whole workflow and the system was suitable for various soybean genotypes, with an average transformation frequency of 58-64%. Higher transformation frequency was observed when wounded cotyledons from 1-day-germination seeds were inoculated and co-cultivated with A. rhizogenes in 1/2 B5 (Gamborg' B-5) medium. The addition of herbicide selection to root production medium increased the transformation frequency to 69%. To test the applicability of the hairy root system for gene functional analysis, we evaluated the protein expression and subcellular localization in transformed hairy roots. Transgenic hairy roots exhibited significantly increased GFP fluorescence and appropriate protein subcellular localization. Protein-protein interactions by BiFC (Bimolecular Fluorescent Complimentary) were also explored using the hairy root system. Fluorescence observations showed that protein interactions could be observed in the root cells. Additionally, hairy root transformation allowed soybean target sgRNA screening for CRISPR/Cas9 gene editing. Therefore, the protocol here enables high-throughput functional characterization of candidate genes in soybean. CONCLUSION: A rapid, simple, and highly efficient A. rhizogenes-mediated hairy root transformation system was established for soybean gene functional analysis, including protein expression, subcellular localization, protein-protein interactions and gene editing system evaluation.

Histidine kinase MHZ1/OsHK1 interacts with ethylene receptors to regulate root growth in rice.

Ethylene plays essential roles during adaptive responses to water-saturating environments in rice, but knowledge of its signaling mechanism remains limited. Here, through an analysis of a rice ethylene-response mutant mhz1, we show that MHZ1 positively modulates root ethylene responses. MHZ1 encodes the rice histidine kinase OsHK1. MHZ1/OsHK1 is autophosphorylated at a conserved histidine residue and can transfer the phosphoryl signal to the response regulator OsRR21 via the phosphotransfer proteins OsAHP1/2. This phosphorelay pathway is required for root ethylene responses. Ethylene receptor OsERS2, via its GAF domain, physically interacts with MHZ1/OsHK1 and inhibits its kinase activity. Genetic analyses suggest that MHZ1/OsHK1 acts at the level of ethylene perception and works together with the OsEIN2-mediated pathway to regulate root growth. Our results suggest that MHZ1/OsHK1 mediates the ethylene response partially independently of OsEIN2, and is directly inhibited by ethylene receptors, thus revealing mechanistic details of ethylene signaling for root growth regulation.

Identification of rice ethylene-response mutants and characterization of MHZ7/OsEIN2 in distinct ethylene response and yield trait regulation.

Ethylene plays essential roles in adaptive growth of rice plants in water-saturating environment; however, ethylene signaling pathway in rice is largely unclear. In this study, we report identification and characterization of ethylene-response mutants based on the specific ethylene-response phenotypes of etiolated rice seedlings, including ethylene-inhibited root growth and ethylene-promoted coleoptile elongation, which is different from the ethylene triple-response phenotype in Arabidopsis. We establish an efficient system for screening and a set of rice mutants have been identified. Genetic analysis reveals that these mutants form eight complementation groups. All the mutants show insensitivity or reduced sensitivity to ethylene in root growth but exhibit differential responses in coleoptile growth. One mutant group mhz7 has insensitivity to ethylene in both root and coleoptile growth. We identified the corresponding gene by a map-based cloning method. MHZ7 encodes a membrane protein homologous to EIN2, a central component of ethylene signaling in Arabidopsis. Upon ethylene treatment, etiolated MHZ7-overexpressing seedlings exhibit enhanced coleoptile elongation, increased mesocotyl growth and extremely twisted short roots, featuring enhanced ethylene-response phenotypes in rice. Grain length was promoted in MHZ7-transgenic plants and 1000-grain weight was reduced in mhz7 mutants. Leaf senescent process was also affected by MHZ7 expression. Manipulation of ethylene signaling may improve adaptive growth and yield-related traits in rice.