Low phosphorus promotes NSP1–NSP2 heterodimerization to enhance strigolactone biosynthesis and regulate shoot and root architecture in rice

Phosphorus is an essential macronutrient for plant development and metabolism,and plants have evolved ingenious mechanisms to overcome phosphate(Pi)starvation.However,the molecular mechanisms underlying the regulation of shoot and root architecture by low phosphorus conditions and the coordinated utilization of Pi and nitrogen remain largely unclear.Here,we show that Nodulation Signaling Pathway 1(NSP1)and NSP2 regulate rice tiller number by promoting the biosynthesis of strigolactones(SLs),a class of phytohormones with fundamental effects on plant architecture and environmental responses.We found that NSP1 and NSP2 are induced by Oryza sativa PHOSPHATE STARVATION RESPONSE2(OsPHR2)in response to low-Pi stress and form a complex to directly bind the promoters of SL biosynthesis genes,thus markedly increasing SL biosynthesis in rice.Interestingly,the NSP1/2–SL signaling module represses the expression of CROWN ROOTLESS 1(CRL1),a newly identified early SL-responsive gene in roots,to restrain lateral root density under Pi deficiency.We also demonstrated that GR24^(4DO) treatment under normal conditions inhibits the expression of OsNRTs and OsAMTs to suppress nitrogen absorption but enhances the expression of OsPTs to promote Pi absorption,thus facilitating the balance between nitrogen and phosphorus uptake in rice.Importantly,we found that NSP1p:NSP1 and NSP2p:NSP2 transgenic plants show improved agronomic traits and grain yield under low-and medium-phosphorus conditions.Taken together,these results revealed a novel regulatory mechanism of SL biosynthesis and signaling in response to Pi starvation,providing genetic resources for improving plant architecture and nutrient-use efficiency in low-Pi environments.

Biological nitrogen fixation in cereal crops:Progress,strategies,and perspectives

Nitrogen is abundant in the atmosphere but is generally the most limiting nutrient for plants.The inability of many crop plants,such as cereals,to directly utilize freely available atmospheric nitrogen gas means that their growth and production often rely heavily on the application of chemical fertilizers,which leads to greenhouse gas emissions and the eutrophication of water.By contrast,legumes gain access to nitrogen through symbiotic association with rhizobia.These bacteria convert nitrogen gas into biologically available ammonia in nodules through a process termed symbiotic biological nitrogen fixation,which plays a decisive role in ecosystem functioning.Engineering cereal crops that can fix nitrogen like legumes or associate with nitrogen-fixing microbiomes could help to avoid the problems caused by the overuse of synthetic nitrogen fertilizer.With the development of synthetic biology,various efforts have been undertaken with the aim of creating so-called‘‘N-self-fertilizing’’crops capable of performing autonomous nitrogen fixation to avoid the need for chemical fertilizers.In this review,we briefly summarize the history and current status of engineering N-self-fertilizing crops.We also propose several potential biotechnological approaches for incorporating biological nitrogen fixation capacity into non-legume plants.

OsCERK1-Mediated Chitin Perception and Immune Signaling Requires Receptor-like Cytoplasmic Kinase 185 to Activate an MAPK Cascade in Rice

Conserved pathogen-associated molecular patterns （PAMPs）, such as chitin, are perceived by pattem recognition receptors （PRRs） located at the host cell surface and trigger rapid activation of mitogen- activated protein kinase （MAPK） cascades, which are required for plant resistance to pathogens. However, the direct links from PAMP perception to MAPK activation in plants remain largely unknown. In this study, we found that the PRR-associated receptor-like cytoplasmic kinase Oryza sativa RLCK185 transmits immune signaling from the PAMP receptor OsCERK1 to an MAPK signaling cascade through interaction with an MAPK kinase kinase, OsMAPKKKε, which is the initial kinase of the MAPK cascade. OsRLCK185 interacts with and phosphorylates the C-terminal regulatory domain of OsMAPKKKε. Coexpression of phosphomi- metic OsR LCK185 and OsMAPKKKε activates MAPK3/6 phosphorylation in Nicotiana benthamiana leaves. Moreover, OsMAPKKKε interacts with and phosphorylates OsMKK4, a key MAPK kinase that transduces the chitin signal. Overexpression of OsMAPKKKε increases chitin-induced MAPK3/6 activation, whereas OsMAPKKKε knockdown compromises chitin-induced MAPK3/6 activation and resistance to rice blast fungus. Taken together, our results suggest the existence of a phospho-signaling pathway from cell surface chitin perception to intraceilular activation of an MAPK cascade in rice.

Medicago AP2-Domain Transcription Factor WRI5a Is a Master Regulator of Lipid Biosynthesis and Transfer during Mycorrhizal Symbiosis

Most land plants have evolved a mutualistic symbiosis with arbuscular mycorrhiza (AM)fungi that improve nutrient acquisition from the soil.In return,up to 20% of host plant photosynthate is transferred to the mycorrhizal fungus in the form of lipids and sugar.Nutrient exchange must be regulated by both partners in order to maintain a reliable symbiotic relationship.However,the mechanisms underlying the regulation of lipid transfer from the plantto the AM fungus remain elusive.Here,we show that the Medicago truncatula AP2/EREBP transcription factor WRI5a,and likely its two homologs WRI5b/Erfl and WRI5c,are master regulators of AM symbiosis Controlling lipid transfer and periarbuscular membrane formation.We found that WRI5a binds AW-box cis-regulatory elements in the promoters of M.truncatula STR,which encodes a periarbuscular membrane-localized ABC transporter required for lipid transfer from the plant to the AM fungus, and MtPT4,whichr encodes a phosphate transporter required for phosphate transfer from the AM fungus to the plant.The hairy roots of the M.truncatula wti5a mutant and RNAi composite plants displayed impaired arbuscule formation,whereas overexpression of WRI5a resulted in enhanced expression of STR and MtPT4,suggesting that WRI5a regulates bidirectional symbiotic nutrient exchange.Moreover,we found that WRI5a and RAM1(Required for Arbuscular Mycorrhization symbiosis 1),which encodes a GRASdomain transcription factor,regulate each other at the transcriptional level,forming a positive feedback loop for regulatingAM symbiosis.Collectively,our data suggest a role for WRI5a in controlling bidirectional nutrient exchange and periarbuscular membrane formation via the regulation of genes involved in the biosynthesis of fatty acids and phosphate uptake in arbuscule-containing cells.

A LysM Receptor Heteromer Mediates Perception of Arbuscular Mycorrhizal Symbiotic Signal in Rice

Symbiotic microorganisms improve nutrient uptake by plants.To initiate mutualistic symbiosis with arbus-cular mycorrhizal(AM)fungi,plants perceive Myc factors,including lipochitooligosaccharides(LCOs)and short-chain chitooligosaccharides(CO4/CO5),secreted by AM fungi.However,the molecular mechanism of Myc factor perception remains elusive.In this study,we identified a heteromer of LysM receptor-like kinases consisting of OsMYR1/OsLYK2 and OsCERK1 that mediates the perception of AM fungi in rice.CO4 directly binds to OsMYR1,promoting the dimerization and phosphorylation of this receptor complex.Compared with control plants,Osmyr1 and Oscerk1 mutant rice plants are less sensitive to Myc factors and show decreased AM colonization.We engineered transgenic rice by expressing chimeric receptors that respectively replaced the ectodomains of OsMYR1 and OsCERK1 with those from the homologous Nod factor receptors MtNFP and MtL YK3 of Medicago truncatula.Transgenic plants displayed increased cal-cium oscillations in response to Nod factors compared with control rice.Our study provides significant mechanistic insights into AM symbiotic signal perception in rice.Expression of chimeric Nod/Myc recep-tors achieves a potentially important step toward generating cereals that host nitrogen-fixing bacteria.

Mycorrhizal symbiosis modulates the rhizosphere microbiota to promote rhizobia–legume symbiosis

Plants establish symbioses with mutualistic fungi,such as arbuscular mycorrhizal(AM)fungi,and bacteria,such as rhizobia,to exchange key nutrients and thrive.Plants and symbionts have coevolved and represent vital components of terrestrial ecosystems.Plants employ an ancestral AM signaling pathway to establish intracellular symbioses,including the legume–rhizobia symbiosis,in their roots.Nevertheless,the relationship between the AM and rhizobial symbioses in native soil is poorly understood.Here,we examined how these distinct symbioses affect root-associated bacterial communities in Medicago truncatula by performing quantitative microbiota profiling(QMP)of 16S rRNA genes.We found that M.truncatula mutants that cannot establish AM or rhizobia symbiosis have an altered microbial load(quantitative abundance)in the rhizosphere and roots,and in particular that AM symbiosis is required to assemble a normal quantitative root-associated microbiota in native soil.Moreover,quantitative microbial co-abundance network analyses revealed that AM symbiosis affects Rhizobiales hubs among plant microbiota and benefits the plant holobiont.Through QMP of rhizobial rpoB and AM fungal SSU rRNA genes,we revealed a new layer of interaction whereby AM symbiosis promotes rhizobia accumulation in the rhizosphere of M.truncatula.We further showed that AM symbiosis-conditioned microbial communities within the M.truncatula rhizosphere could promote nodulation in different legume plants in native soil.Given that the AM and rhizobial symbioses are critical for crop growth,our findings might inform strategies to improve agricultural management.Moreover,our work sheds light on the co-evolution of these intracellular symbioses during plant adaptation to native soil conditions.

Hormone modulation of legume-rhizobial symbiosis

Leguminous plants can establish symbiotic associations with diazotropic rhizobia to form nitrogen- fixating nodules, which are classified as determinate or indeterminate based on the persistence of nodule meristem. The formation of nitrogen-fixing nodules requires coordinating rhizobial infection and root nodule organogenesis. The formation of an infection thread and the extent of nodule formation are largely under plant control, but vary with environmental conditions and the physiological state of the host plants. Many achievements in these two areas have been made in recent decades. Phytohormone signaling pathways have gradually emerged as important regulators of root nodule symbio- sis. Cytokinin, strigolactones （SLs） and local accumulation of auxin can promote nodule development. Ethylene, jasmonic acid （JA）, abscisic acid （ABA） and gibberellic acid （GA） all negatively regulate infection thread formation and nodule development. However, salicylic acid （SA） and brassinosteroids （BRs） have different effects on the formation of these two nodule types. Some peptide hormones are also involved in nodulation. This review summarizes recent findings on the roles of these plant hormones in legume-rhizobial symbiosis, and we propose that DELLA proteins may function as a node to integrate plant hormones to regulate nodulation.

Mechanisms underlying legume-rhizobium symbioses

Legumes,unlike most land plants,can form symbiotic root nodules with nitrogen-fixing bacteria to secure nitrogen for growth.The formation of nitrogen-fixing nodules on legume roots requires the coordination of rhizobial infection at the root epidermis with cell division in the cortex.The nodules house the nitrogen-fixing rhizobia in organelle-like structures known as symbiosomes,which enable nitrogen fixation and facilitate the exchange of metabolites between the host and symbionts.In addition to this beneficial interaction,legumes are continuously exposed to would-be pathogenic microbes;therefore the ability to discriminate pathogens from symbionts is a major determinant of plant survival under natural conditions.Here,we summarize recent advances in the understanding of root nodule symbiosis signaling,transcriptional regulation,and regulation of plant immunity during legume-rhizobium symbiosis.In addition,we propose several important questions to be addressed and provide insights into the potential for engineering the capacity to fix nitrogen in legume and nonlegume plants.

Phosphorylation of MtRopGEF2 by LYK3 mediates MtROP activity to regulate rhizobial infection in Medicago truncatula

The formation of nitrogen-fixing no dules on legume roots requires the coordination of infection by rhizobia at the root epidermis with the initiation of cell divisions in the root cortex.During infection,rhizobia attach to the tip of elongating root hairs which then curl to entrap the rhizobia.However,the mechanism of root hair deformation and curling in response to symbiotic signals is still elusive.Here,we found that small GTPases(MtRac1/MtROP9 and its homologs)are required for root hair development and rhizobial infection in Medicago truncatula.Our results show that the Nod factor receptor LYK3 phosphorylates the guanine nucleotide exchange factor MtRopGEF2 at S73 which is critical for the polar growth of root hairs.In turn,phosphorylated MtRopGEF2 can activate MtRac1.Activated MtRac1 was found to localize at the tips of root hairs and to strongly interact with LYK3 and NFP.Taken together,our results support the hypothesis that MtRac1,LYK3,and NFP form a polarly localized receptor complex that regulates root hair deformation during rhizobial infection.

Arbuscular mycorrhizal associations and the major regulators

Plants growing in natural soils encounter diverse biotic and abiotic stresses and have adapted with sophisticated strategies to deal with complex environments such as changing root system structure,evoking biochem-ical responses and recruiting microbial partners.Under selection pressure,plants and their associated microorgan-isms assemble into a functional entity known as a holobiont.The commonest cooperative interaction is between plant roots and arbuscular mycorrhizal(AM)fungi.About 80%of terrestrial plants can form AM symbiosis with the ancient phylum Glomeromycota.A very large network of extraradical and intraradical mycelium of AM fungi connects the underground biota and the nearby carbon and nutrient fluxes.Here,we discuss recent progress on the regulators of AM associations with plants,AM fungi and their surrounding environments,and explore further mechanistic insights.

The chromosome-level genome assembly of Astragalus sinicus and comparative genomic analyses provide new resources and insights for understanding legume-rhizobial interactions

The legume species Astragalus sinicus(Chinese milk vetch[CMV])has been widely cultivated for centuries in southern China as one of the most important green manures/cover crops for improving rice productivity and preventing soil degeneration.In this study,we generated the first chromosome-scale reference genome of CMV by combining PacBio and Illumina sequencing with high-throughput chromatin conformation capture(Hi-C)technology.The CMV genome was 595.52 Mb in length,with a contig N50 size of 1.50 Mb.Long terminal repeats(LTRs)had been amplified and contributed to genome size expansion in CMV.CMV has undergone two whole-genome duplication(WGD)events,and the genes retained after the WGD shared by Papilionoideae species shaped the rhizobial symbiosis and the hormonal regulation of nodulation.The chalcone synthase(CHS)gene family was expanded and was expressed primarily in the roots of CMV.Intriguingly,we found that resistance genes were more highly expressed in roots than in nodules of legume species,suggesting that their expression may be increased to bolster plant immunity in roots to cope with pathogen infection in legumes.Our work sheds light on the genetic basis of nodulation and symbiosis in CMV and provides a benchmark for accelerating genetic research and molecular breeding in the future.