Efficient genome editing in rice with miniature Cas12f variants.

Genome editing, particularly using the CRISPR/Cas system, has revolutionized biological research and crop improvement. Despite the widespread use of CRISPR/Cas9, it faces limitations such as PAM sequence requirements and challenges in delivering its large protein into plant cells. The hypercompact Cas12f, derived from Acidibacillus sulfuroxidans (AsCas12f), stands out due to its small size of only 422 amino acids and its preference for a T-rich motif, presenting advantageous features over SpCas9. However, its editing efficiency is extremely low in plants. Recent studies have generated two AsCas12f variants, AsCas12f-YHAM and AsCas12f-HKRA, demonstrating higher editing efficiencies in mammalian cells, yet their performance in plants remains unexplored. In this study, through a systematic investigation of genome cleavage activity in rice, we unveiled a substantial enhancement in editing efficiency for both AsCas12f variants, particularly for AsCas12f-HKRA, which achieved an editing efficiency of up to 53%. Furthermore, our analysis revealed that AsCas12f predominantly induces deletion in the target DNA, displaying a unique deletion pattern primarily concentrated at positions 12, 13, 23, and 24, resulting in deletion size mainly of 10 and 11 bp, suggesting significant potential for targeted DNA deletion using AsCas12f. These findings expand the toolbox for efficient genome editing in plants, offering promising prospects for precise genetic modifications in agriculture. Supplementary Information: The online version contains supplementary material available at 10.1007/s42994-024-00168-2.

Rice floury endosperm26 encoding a mitochondrial single-stranded DNA-binding protein is essential for RNA-splicing of mitochondrial genes and endosperm development.

Endosperm, the major storage organ in cereal grains, determines the grain yield and quality. Mitochondria provide the energy for dry matter accumulation, in the endosperm development. Although mitochondrial single-stranded DNA-binding proteins (mtSSBs) play a canonical role in the maintenance of single-stranded mitochondrial DNA, their molecular functions in RNA processing and endosperm development remain obscure. Here, we report a defective rice endosperm mutant, floury endosperm26 (flo26), which develops abnormal starch grains in the endosperm. Map-based cloning and complementation experiments showed that FLO26 allele encodes a mitochondrial single-stranded DNA-binding protein, named as mtSSB1.1. Loss of function of mtSSB1.1 affects the transcriptional level of many mitochondrially-encoded genes and RNA splicing of nad1, a core component of respiratory chain complex I in mitochondria. As a result, dysfunctional mature nad1 led to dramatically decreased complex I activity, thereby reducing ATP production. Our results reveal that mtSSB1.1 plays an important role in the maintenance of mitochondrial function and endosperm development by stabilizing the splicing of mitochondrial RNA in rice.

A mixed blessing of influent leachate microbes in downstream biotreatment systems of a full-scale landfill leachate treatment plant.

In landfill leachate treatment plants (LLTPs), the microbiome plays a pivotal role in the decomposition of organic compounds, reduction in nutrient levels, and elimination of toxins. However, the effects of microbes in landfill leachate influents on downstream treatment systems remain poorly understood. To address this knowledge gap, we collected 23 metagenomic and 12 metatranscriptomic samples from landfill leachate and activated sludge from various treatment units in a full-scale LLTP. We successfully recovered 1,152 non-redundant metagenome-assembled genomes (MAGs), encompassing a wide taxonomic range, including 48 phyla, 95 classes, 166 orders, 247 families, 238 genera, and 1,152 species. More diverse microbes were observed in the influent leachate than in the downstream biotreatment systems, among which, an unprecedented ∼30 % of microbes with transcriptional expression migrated from the influent to the biological treatment units. Network analysis revealed that 399 shared MAGs across the four units exhibited high node centrality and degree, thus supporting enhanced interactions and increased stability of microbial communities. Functional reconstruction and genome characterization of MAGs indicated that these shared MAGs possessed greater capabilities for carbon, nitrogen, sulfur, and arsenic metabolism compared to non-shared MAGs. We further identified a novel species of Zixibacteria in the leachate influent with discrete lineages from those in other environments that accounted for up to 17 % of the abundance of the shared microbial community and exhibited notable metabolic versatility. Meanwhile, we presented groundbreaking evidence of the involvement of Zixibacteria-encoded genes in the production of harmful gas emissions, such as N2O and H2S, at the transcriptional level, thus suggesting that influent microbes may pose safety risks to downstream treatment systems. In summary, this study revealed the complex impact of the influent microbiome on LLTP and emphasizes the need to consider these microbial characteristics when designing treatment technologies and strategies for landfill leachate management.

Transport dynamics of watershed discharged diffuse phosphorus pollution load to the lake in middle of Yangtze River Basin.

Diffuse pollution, including that in the lower and middle reaches of the Yangtze River, is the primary source of pollution in several agricultural watersheds globally. As the largest river basin in China, the Yangtze River Basin has suffered from total phosphorus (TP) pollution in the past decade owing to diffuse pollution and aquatic ecology destruction, especially in the midstream tributaries and mid-lower reaches of the lakes. However, the transport dynamics of diffuse pollutants, such as phosphorus (P) from land to water bodies have not been well evaluated, which is of great significance for quantifying nutrient loss and its impact on water bodies. In this study, diffuse pollution estimation with remote sensing (DPeRS) model coupled with Soil and Water Assessment Tools (SWAT) was utilized to simulate the transport dynamics of P, investigate the spatial heterogeneity and P sources in the Poyang Lake Basin. Additionally, the P transport mechanism from land to water and the migration process in water bodies were considered to investigate the impact of each loss unit on the water body and evaluate the load generated by diverse pollution types. The estimated diffuse TP loss was 6016 t P·yr-1, and the load to inflow rivers and to Poyang Lake were 11,619 and 9812 t P·yr-1, respectively. Gan River Basin (51.09%) contributed most TP to Poyang Lake among five inflow rivers, while waterfront area demonstrated the highest TP load per unit area with 0.057 t km-2·yr-1. Our study also identified P sources in the sub-basins and emphasized agricultural diffuse sources, especially planting, as the most significant factor contributing to TP pollution. Additionally, to improve the aquatic environment and water ecological conditions, further nutrient management should be applied using a comprehensive approach that encompasses the entire process, from source transportation to the water body.

Metagenomic analysis unveils the underexplored roles of prokaryotic viruses in a full-scale landfill leachate treatment plant.

Enormous viral populations have been identified in activated sludge systems, but their ecological and biochemical roles in landfill leachate treatment plants remain poorly understood. To address this knowledge gap, we conducted an in-depth analysis using 36 metagenomic datasets that we collected and sequenced during a half-year time-series sampling campaign at six sites in a full-scale landfill leachate treatment plant (LLTP), elucidating viral distribution, virus‒host dynamics, virus-encoded auxiliary metabolic genes (AMGs), and viral contributions to the spread of virulence and antibiotic resistance genes. Our findings demonstrated that viral and prokaryotic communities differed widely among different treatment units, with stability over time. LLTP viruses were linked to various prokaryotic hosts, spanning 35 bacterial phyla and one archaeal phylum, which included the core microbes involved in biological treatments, as well as some of the less well-characterized microbial dark matter phyla. By encoding 2364 auxiliary metabolic genes (AMGs), viruses harbored the potential to regulate microbial nucleotide metabolism, facilitate the biodegradation of complex organic matter, and enhance flocculation and settling in biological treatment plants. The abundance distribution of AMGs varied considerably across treatment units and showed a lifestyle-dependent pattern with temperate virus-associated AMGs exhibiting a higher average abundance in downstream biological treatment units and effluent water. Meanwhile, temperate viruses tended to carry a higher load of virulence factor genes (VFGs), antibiotic resistance genes (ARGs), and biotic and metal resistance genes (BMRGs), and engaged in more frequent gene exchanges with prokaryotes than lytic viruses, thus acting as a pivotal contributor to the dissemination of pathogenicity and resistance genes in downstream LLTP units. This study provided a comprehensive profile of viral and prokaryotic communities in the LLTP and unveiled the varying roles of different-lifestyle viruses in biochemical processes and water quality safety.

The transcriptional hub SHORT INTERNODES1 integrates hormone signals to orchestrate rice growth and development.

Plants have evolved sophisticated mechanisms to coordinate their growth and stress responses via integrating various phytohormone signaling pathways. However, the precise molecular mechanisms orchestrating integration of the phytohormone signaling pathways remain largely obscure. In this study, we found that the rice (Oryza sativa) short internodes1 (shi1) mutant exhibits typical auxin-deficient root development and gravitropic response, brassinosteroid (BR)-deficient plant architecture and grain size as well as enhanced abscisic acid (ABA)-mediated drought tolerance. Additionally, we found that the shi1 mutant is also hyposensitive to auxin and BR treatment but hypersensitive to ABA. Further, we showed that OsSHI1 promotes the biosynthesis of auxin and BR by activating the expression of OsYUCCAs and D11, meanwhile dampens ABA signaling by inducing the expression of OsNAC2, which encodes a repressor of ABA signaling. Furthermore, we demonstrated that 3 classes of transcription factors, AUXIN RESPONSE FACTOR 19 (OsARF19), LEAF AND TILLER ANGLE INCREASED CONTROLLER (LIC), and OsZIP26 and OsZIP86, directly bind to the promoter of OsSHI1 and regulate its expression in response to auxin, BR, and ABA, respectively. Collectively, our results unravel an OsSHI1-centered transcriptional regulatory hub that orchestrates the integration and self-feedback regulation of multiple phytohormone signaling pathways to coordinate plant growth and stress adaptation.

Fructose-6-phosphate-2-kinase/fructose-2,6-bisphosphatase regulates energy metabolism and synthesis of storage products in developing rice endosperm.

Starch accounts for about 80-85 % of the dry weight of grains and determines yield by impact on grain weight. And, the content and composition of starch also determine appearance, eating, cooking and nutritional quality of rice. By coordinating crucial reactions of the primary carbohydrate metabolism in all eukaryotes, fructose-2,6-bisphosphate (Fru-2,6-P2) is a traffic signal in metabolism. However, the metabolic regulation of starch in plant sink tissues by Fru-2,6-P2 remains unclear. Here we isolated rice mutant floury endosperm23 (flo23) which has opaque endosperm and anomalous compound starch grains (SGs). flo23 mutant grains had reduced contents of starch, lipids and proteins. Map-based cloning and genetic complementation experiments showed that FLO23 encodes a cytoplasmic Fructose-6-phosphate-2-kinase/Fructose-2,6-bisphosphatase (F2KP). Mutation of OsF2KP2 decreased Fru-2,6-P2 content in endosperm cells, leading to drastically reduced phosphoenolpyruvate (PEP) and pyruvate contents and disordered glycolysis and energy metabolism. The results imply that OsF2KP2 participates in the glycolytic pathway by providing precursors and energy for synthesis of grain storage compounds.

Rice FLOURY ENDOSPERM22, encoding a pentatricopeptide repeat protein, is involved in both mitochondrial RNA splicing and editing and is crucial for endosperm development.

Most of the reported P-type pentatricopeptide repeat (PPR) proteins play roles in organelle RNA stabilization and splicing. However, P-type PPRs involved in both RNA splicing and editing have rarely been reported, and their underlying mechanism remains largely unknown. Here, we report a rice floury endosperm22 (flo22) mutant with delayed amyloplast development in endosperm cells. Map-based cloning and complementation tests demonstrated that FLO22 encodes a mitochondrion-localized P-type PPR protein. Mutation of FLO22 resulting in defective trans-splicing of mitochondrial nad1 intron 1 and perhaps causing instability of mature transcripts affected assembly and activity of complex Ⅰ, and mitochondrial morphology and function. RNA-seq analysis showed that expression levels of many genes involved in starch and sucrose metabolism were significantly down-regulated in the flo22 mutant compared with the wild type, whereas genes related to oxidative phosphorylation and the tricarboxylic acid cycle were significantly up-regulated. In addition to involvement in splicing as a P-type PPR protein, we found that FLO22 interacted with DYW3, a DYW-type PPR protein, and they may function synergistically in mitochondrial RNA editing. The present work indicated that FLO22 plays an important role in endosperm development and plant growth by participating in nad1 maturation and multi-site editing of mitochondrial messager RNA.

Plastidic pyruvate dehydrogenase complex E1 component subunit Alpha1 is involved in galactolipid biosynthesis required for amyloplast development in rice.

Starch accounts for over 80% of the total dry weight in cereal endosperm and determines the kernel texture and nutritional quality. Amyloplasts, terminally differentiated plastids, are responsible for starch biosynthesis and storage. We screened a series of rice mutants with floury endosperm to clarify the mechanism underlying amyloplast development and starch synthesis. We identified the floury endosperm19 (flo19) mutant which shows opaque of the interior endosperm. Abnormal compound starch grains (SGs) were present in the endosperm cells of the mutant. Molecular cloning revealed that the FLO19 allele encodes a plastid-localized pyruvate dehydrogenase complex E1 component subunit α1 (ptPDC-E1-α1) that is expressed in all rice tissues. In vivo enzyme assays demonstrated that the flo19 mutant showed decreased activity of the plastidic pyruvate dehydrogenase complex. In addition, the amounts of monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG) were much lower in the developing flo19 mutant endosperm, suggesting that FLO19 participates in fatty acid supply for galactolipid biosynthesis in amyloplasts. FLO19 overexpression significantly increased seed size and weight, but did not affect other important agronomic traits, such as panicle length, tiller number and seed setting rate. An analysis of single nucleotide polymorphism data from a panel of rice accessions identified that the pFLO19L haplotype was positively associated with grain length, implying a potential application in rice breeding. In summary, our study demonstrates that FLO19 is involved in galactolipid biosynthesis which is essential for amyloplast development and starch biosynthesis in rice.

Subunit E isoform 1 of vacuolar H+-ATPase OsVHA enables post-Golgi trafficking of rice seed storage proteins.

Dense vesicles (DVs) are Golgi-derived plant-specific carriers that mediate post-Golgi transport of seed storage proteins in angiosperms. How this process is regulated remains elusive. Here, we report a rice (Oryza sativa) mutant, named glutelin precursor accumulation8 (gpa8) that abnormally accumulates 57-kDa proglutelins in the mature endosperm. Cytological analyses of the gpa8 mutant revealed that proglutelin-containing DVs were mistargeted to the apoplast forming electron-dense aggregates and paramural bodies in developing endosperm cells. Differing from previously reported gpa mutants with post-Golgi trafficking defects, the gpa8 mutant showed bent Golgi bodies, defective trans-Golgi network (TGN), and enlarged DVs, suggesting a specific role of GPA8 in DV biogenesis. We demonstrated that GPA8 encodes a subunit E isoform 1 of vacuolar H+-ATPase (OsVHA-E1) that mainly localizes to TGN and the tonoplast. Further analysis revealed that the luminal pH of the TGN and vacuole is dramatically increased in the gpa8 mutant. Moreover, the colocalization of GPA1 and GPA3 with TGN marker protein in gpa8 protoplasts was obviously decreased. Our data indicated that OsVHA-E1 is involved in endomembrane luminal pH homeostasis, as well as maintenance of Golgi morphology and TGN required for DV biogenesis and subsequent protein trafficking in rice endosperm cells.

BRITTLE PLANT1 is required for normal cell wall composition and mechanical strength in rice.

A series of nucleotide sugar interconversion enzymes (NSEs) generate the activated sugar donors required for biosynthesis of cell wall matrix polysaccharides and glycoproteins. UDP-glucose 4-epimerases (UGEs) are NSEs that function in the interconversion of UDP-glucose (UDP-Glc) and UDP-galactose (UDP-Gal). The roles of UDP-glucose 4-epimerases in monocots remain unclear due to redundancy in the pathways. Here, we report a brittle plant (bp1) rice mutant that exhibits brittle leaves and culms at all growth stages. The mutant culms had reduced levels of rhamnogalacturonan I, homogalacturonan, and arabinogalactan proteins. Moreover, the mutant had altered contents of uronic acids, neutral noncellulosic monosaccharides, and cellulose. Map-based cloning demonstrated that OsBP1 encodes a UDP-glucose 4-epimerase (OsUGE2), a cytosolic protein. We also show that BP1 can form homo- and hetero-protein complexes with other UGE family members and with UDP-galactose transporters 2 (OsUGT2) and 3 (OsUGT3), which may facilitate the channeling of Gal to polysaccharides and proteoglycans. Our results demonstrate that BP1 participates in regulating the sugar composition and structure of rice cell walls.

The miR156/SPL module regulates apple salt stress tolerance by activating MdWRKY100 expression.

Salt stress dramatically impedes plant growth and development as well as crop yield. The apple production regions are reduced every year, because of the secondary salt damage by improper fertilization and irrigation. To expand the cultivation area of apple (Malus domestica) and select salt-resistant varieties, the mechanism of salt tolerance in apple is necessary to be clarified. The miR156/SPL regulatory module plays key roles in embryogenesis, morphogenesis, life cycle stage transformation, flower formation and other processes. However, its roles in the mechanisms of salt tolerance are unknown. In order to elucidate the mechanism of 156/SPL regulating salt stress in apple, we performed RLM-5' RACE and stable genetic transformation technology to verify that both mdm-MIR156a and MdSPL13 responded to salt stress in apple and that the latter was the target of the former. MIR156a overexpression weakened salt resistance in apple whereas MdSPL13 overexpression strengthened it. A total of 6094 differentially expressed genes relative to nontransgenic apple plants were found by RNA-Seq analysis of MdSPL13OE. Further verification indicated that MdSPL13 targeted the MdWRKY100 gene promoter. Moreover, MdWRKY100 overexpression enhanced salt tolerance in apple. Our results revealed that the miR156/SPL module regulates salt tolerance by up-regulating MdWRKY100 in apple. This study is the first to elucidate the mechanism underlying the miRNA network response to salt stress in apple and provides theoretical and empirical bases and genetic resources for the molecular breeding of salt tolerance in apple.

white panicle2 encoding thioredoxin z, regulates plastid RNA editing by interacting with multiple organellar RNA editing factors in rice.

Thioredoxins (TRXs) occur in plant chloroplasts as complex disulphide oxidoreductases. Although many biological processes are regulated by thioredoxins, the regulatory mechanism of chloroplast TRXs are largely unknown. Here we report a rice white panicle2 mutant caused by a mutation in the thioredoxin z gene, an orthologue of AtTRX z in Arabidopsis. white panicle2 (wp2) seedlings exhibited a high-temperature-sensitive albinic phenotype. We found that plastid multiple organellar RNA editing factors (MORFs) were the regulatory targets of thioredoxin z. We showed that OsTRX z protein physically interacts with OsMORFs in a redox-dependent manner and that the redox state of a conserved cysteine in the MORF box is essential for MORF-MORF interactions. wp2 and OsTRX z knockout lines show reduced editing efficiencies in many plastidial-encoded genes especially under high-temperature conditions. An Arabidopsis trx z mutant also exhibited significantly reduced chloroplast RNA editing. Our combined results suggest that thioredoxin z regulates chloroplast RNA editing in plants by controlling the redox state of MORFs.

A Novel Ourmia-Like Mycovirus Confers Hypovirulence-Associated Traits on Fusarium oxysporum.

Fusarium wilt caused by Fusarium oxysporum f. sp. momordicae (FoM) is an important fungal disease that affects the production of bitter gourd. Hypovirulence-associated mycoviruses have great potential and application prospects for controlling the fungal disease. In this study, a novel ourmia-like virus, named Fusarium oxysporum ourmia-like virus 1 (FoOuLV1), was isolated from FoM strain HuN8. The viral genomic RNA is 2,712 nucleotides (nt) in length and contains an open reading frame (ORF) encoding a putative RNA-dependent RNA polymerase (RdRp) using either standard or mitochondrial codes. In strain HuN8, there was also a FoOuLV1-associated RNA segment with 1,173 nt in length with no sequence homology. Phylogenetic analysis showed that FoOuLV1 is a member of the genus Magoulivirus of the family Botourmiaviridae. FoOuLV1 was found to be associated with hypovirulence in FoM. Moreover, FoOuLV1 and its hypovirulence trait can be transmitted horizontally to other FoM strains and also to other formae speciale strains of F. oxysporum. In addition, FoOuLV1 showed significant biological control effect against the bitter gourd Fusarium wilt. To our knowledge, this study reveals the first description of a hypovirulence-associated ourmia-like mycovirus, which has the potential to the biological control of Fusarium wilt.

Salvia bowleyana Dunn root is a novel source of salvianolic acid B and displays antitumor effects against gastric cancer cells.

Salvianolic acid B (Sal-B) is widely used in China for the treatment of numerous diseases. Currently, Salvia miltiorrhiza Bunge is the main source of this compound, but Salvia bowleyana Dunn, a surrogate of S. miltiorrhiza Bge, may provide a novel source for obtaining more Sal-B. In the present study, a simple method for separation and purification of phenolic compounds from S. bowleyana Dunn roots was employed. Sal-B was subsequently purified and its inhibitory effect on the gastric cancer HGC-27 and AGS cell lines was investigated. Sal-B extracted from S. bowleyana Dunn displayed significant antitumor activity in proliferation and apoptosis assays. Overall, it was found that S. bowleyana Dunn has a higher Sal-B content than S. miltiorrhiza Bge and may be used as a novel source of this potential anti-gastric cancer compound.

Genome-wide associated study identifies NAC42-activated nitrate transporter conferring high nitrogen use efficiency in rice.

Over-application of nitrogen fertilizer in fields has had a negative impact on both environment and human health. Domesticated rice varieties with high nitrogen use efficiency (NUE) reduce fertilizer for sustainable agriculture. Here, we perform genome-wide association analysis of a diverse rice population displaying extreme nitrogen-related phenotypes over three successive years in the field, and identify an elite haplotype of nitrate transporter OsNPF6.1HapB that enhances nitrate uptake and confers high NUE by increasing yield under low nitrogen supply. OsNPF6.1HapB differs in both the protein and promoter element with natural variations, which are differentially trans-activated by OsNAC42, a NUE-related transcription factor. The rare natural allele OsNPF6.1HapB, derived from variation in wild rice and selected for enhancing both NUE and yield, has been lost in 90.3% of rice varieties due to the increased application of fertilizer. Our discovery highlights this NAC42-NPF6.1 signaling cascade as a strategy for high NUE and yield breeding in rice.

Total Astragalus saponins attenuates CVB3-induced viral myocarditis through inhibiting expression of tumor necrosis factor α and Fas ligand.

BACKGROUND: To investigate the therapeutic effect of total Astragalus saponins (AST) against viral myocarditis in animal and cell models. METHODS: Primary myocardiocytes (PMCs) were stimulated by the coxsackie B (CVB) 3 virus to prepare the cell model of viral myocarditis. Cell viability, apoptosis and the mRNA expression of C-Myc, tumor necrosis factor (TNF)-α and Fas were detected to evaluate the protective effects of AST on CVB3-induced PMC damage. RESULTS: AST could significantly increase survival and decrease the ratio of heart weight: body weight (P<0.05). The level of myocardial fibrosis in the AST group was significantly lower than that in the CVB3 group. Compared with the CVB3 group, the ejection fraction was increased significantly in the AST group. Levels of lactate dehydrogenase and creatine kinase-MB in the peripheral blood of the AST group were significantly lower than those in the control group. In vitro, AST could significantly decrease CVB3-induced PMC apoptosis. Expression of C-Myc, TNF-α, Fas in the AST group was significantly lower than that in the CVB3 group. CONCLUSIONS: It is demonstrated that AST was protective against CVB3-induced viral myocarditis, which may be associated with a decrease in CVB3-induced apoptosis and down-regulation of expression of C-Myc, TNF-α and Fas.

OsNHX5-mediated pH homeostasis is required for post-Golgi trafficking of seed storage proteins in rice endosperm cells.

BACKGROUND: As the major storage protein in rice seeds, glutelins are synthesized at the endoplasmic reticulum (ER) as proglutelins and transported to protein storage vacuoles (PSVs) called PBIIs (Protein body IIs), where they are cleaved into mature forms by the vacuolar processing enzymes. However, the molecular mechanisms underlying glutelin trafficking are largely unknown. RESULTS: In this study, we report a rice mutant, named glutelin precursor accumulation6 (gpa6), which abnormally accumulates massive proglutelins. Cytological analyses revealed that in gpa6 endosperm cells, proglutelins were mis-sorted, leading to the presence of dense vesicles (DVs) and the formation paramural bodies (PMBs) at the apoplast, consequently, smaller PBII were observed. Mutated gene in gpa6 was found to encode a Na+/H+ antiporter, OsNHX5. OsNHX5 is expressed in all tissues analyzed, and its expression level is much higher than its closest paralog OsNHX6. The OsNHX5 protein colocalizes to the Golgi, the trans-Golgi network (TGN) and the pre-vacuolar compartment (PVC) in tobacco leaf epidermal cells. In vivo pH measurements indicated that the lumens of Golgi, TGN and PVC became more acidic in gpa6. CONCLUSIONS: Our results demonstrated an important role of OsNHX5 in regulating endomembrane luminal pH, which is essential for seed storage protein trafficking in rice.

MdWRKY100 encodes a group I WRKY transcription factor in Malus domestica that positively regulates resistance to Colletotrichum gloeosporioides infection.

Apple (Malus domestica) is an important fruit worldwide; however, the development of the apple industry is limited by fungal disease. Apple bitter rot caused by the pathogen Colletotrichum gloeosporioides is one of the most devastating apple diseases, leading to large-scale losses in apple quality and production. WRKY transcription factors have important functions in the regulation of biotic and abiotic stresses. However, their biological and molecular functions in non-model plants, including apple, remain poorly understood. Here, we isolated MdWRKY100 from 'Hanfu' apple. The MdWRKY100 protein fused to green fluorescent protein localized to the nucleus, and MdWRKY100 in yeast cells displayed transcriptional activation activity, which is consistent with the function of a transcription factor. Additionally, several putative cis-acting elements involved in abiotic stress responsiveness were also identified in the MdWRKY100 promoter. Transcriptional analysis revealed that MdWRKY100 was expressed ubiquitously in all examined apple organs. Overexpression in apple increased resistance to Colletotrichum gloeosporioides, while RNAi silencing transgenic plants were more sensitive to Colletotrichum gloeosporioides. Collectively, our data demonstrate that MdWRKY100 is a positive regulator of Colletotrichum gloeosporioides resistance in apple.