Effector Cs02526 from Ciboria shiraiana induces cell death and modulates plant immunity.

Sclerotinia disease is one of the most devastating fungal diseases worldwide, as it reduces the yields of many economically important crops. Pathogen-secreted effectors play crucial roles in infection processes. However, key effectors of Ciboria shiraiana, the pathogen primarily responsible for sclerotinia disease in mulberry (Morus spp.), remain poorly understood. In this study, we identified and functionally characterized the effector Cs02526 in C. shiraiana and found that Cs02526 could induce cell deathin a variety of plants. Moreover, Cs02526-induced cell death was mediated by the central immune regulator BRASSINOSTEROID INSENSITIVE 1-associated receptor kinase 1 (BAK1), dependent on a 67-amino acid fragment. Notably, Cs02526 homologues were widely distributed in hemibiotrophic and necrotrophic phytopathogenic fungi, but the homologues failed to induce cell death in plants. Pre-treatment of plants with recombinant Cs02526 protein enhanced resistance against both C. shiraiana and Sclerotinia sclerotiorum. Furthermore, the pathogenicity of C. shiraiana was diminished upon spraying plants with synthetic dsRNA-Cs02526. In conclusion, our findings highlight the cell death-inducing effector Cs02526 as a potential target for future biological control strategies against plant diseases.

Genome Sequencing of Ciboria shiraiana Provides Insights into the Pathogenic Mechanisms of Hypertrophy Sorosis Scleroteniosis.

Ciboria shiraiana causes hypertrophy sorosis scleroteniosis in mulberry trees, resulting in huge economic losses, and exploring its pathogenic mechanism at a genomic level is important for developing new control methods. Here, genome sequencing of C. shiraiana based on PacBio RSII and Illumina HiSeq 2500 platform as well as manual gap filling was performed. Synteny analysis with Sclerotinia sclerotiorum revealed 16 putative chromosomes corresponding to 16 chromosomes of C. shiraiana. Screening of rapid-evolution genes revealed that 97 and 2.4% of genes had undergone purifying selection and positive selection, respectively. When compared with S. sclerotiorum, fewer secreted effector proteins were found in C. shiraiana. The number of genes involved in pathogenicity, including secondary metabolites, carbohydrate active enzymes, and P450s, in the C. shiraiana genome was comparable with that of other necrotrophs but higher than that of biotrophs and saprotrophs. The growth-related genes and plant cell-wall-degradation-related genes in C. shiraiana were expressed in different developmental and infection stages, and may be potential targets for prevention and control of this pathogen. These results provide new insights into C. shiraiana pathogenic mechanisms, especially host range and necrotrophy features, and lay the foundation for further study of the underlying molecular mechanisms.[Formula: see text] The author(s) have dedicated the work to the public domain under the Creative Commons CC0 "No Rights Reserved" license by waiving all of his or her rights to the work worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.

A meta-analysis of transcriptomic profiles reveals molecular pathways response to cadmium stress of Gramineae.

As a non-essential heavy metal, cadmium (Cd) is toxic to plants. In the last 15 years, over 70 transcriptome studies have been published to decipher the molecular response mechanism against Cd stress in different plants. To extract generalization results from transcriptomic data across different plants and obtain some hub genes that respond to Cd stress, we carried out a meta-analysis of 32 published datasets. Cluster analysis revealed that plant species played a more decisive role than the media used and exposure time in the transcriptome patterns of plant roots response to Cd. The datasets from a Gramineae-like (GL) group were closer in clustering. 838 DEGs were commonly Cd-regulated in at least nine of 18 GL datasets. Gene ontology and KEGG pathway analyses revealed that oxidative stress-related terms and lignin synthesis-related terms were significantly enriched. Mapman analysis revealed that these common DEGs were mainly involved in regulation, cellular response, secondary metabolism, transport, cell wall and lipid metabolism. In Oryza sativa, 15 DEGs were up-regulated in at least four of five HM (As, Cr, Cd, Hg and Pb) groups, such as Os10g0517500 (methionine gamma-lyase) and Os01g0159800 (bHLH107). Moreover, our datasets can be used to retrieve log2FC value of specific genes across 29 studies (48 datasets), which provides data reference for the subsequent selection of HM-related genes. Our results provide the basis for further understanding of Cd tolerance mechanisms in plants.

New Insights into the Structure-Function Relationship of the Endosomal-Type Na+, K+/H+ Antiporter NHX6 from Mulberry (Morus notabilis).

The endosomal-type Na+, K+/H+ antiporters (NHXs) play important roles in K+, vesicle pH homeostasis, and protein trafficking in plant. However, the structure governing ion transport mechanism and the key residues related to the structure-function of the endosomal-type NHXs remain unclear. Here, the structure-function relationship of the only endosomal-type NHX from mulberry, MnNHX6, was investigated by homology modeling, mutagenesis, and localization analyses in yeast. The ectopic expression of MnNHX6 in arabidopsis and Nhx1 mutant yeast can enhance their salt tolerance. MnNHX6's three-dimensional structure, established by homology modeling, was supported by empirical, phylogenetic, and experimental data. Structure analysis showed that MnNHX6 contains unusual 13 transmembrane helices, but the structural core formed by TM5-TM12 assembly is conserved. Localization analysis showed that MnNHX6 has the same endosomal localization as yeast Nhx1/VPS44, and Arg402 is important for protein stability of MnNHX6. Mutagenesis analysis demonstrated MnNHX6 contains a conserved cation binding mechanism and a similar charge-compensated pattern as NHE1, but shares a different role in ion selectivity than the vacuolar-type NHXs. These results improve our understanding of the role played by the structure-function related key residues of the plant endosomal-type NHXs, and provide a basis for the ion transport mechanism study of endosomal-type NHXs.

Ectopic Expression of Mulberry G-Proteins Alters Drought and Salt Stress Tolerance in Tobacco.

Heterotrimeric guanine-nucleotide-binding proteins (G-proteins) play key roles in responses to various abiotic stress responses and tolerance in plants. However, the detailed mechanisms behind these roles remain unclear. Mulberry (Morus alba L.) can adapt to adverse abiotic stress conditions; however, little is known regarding the associated molecular mechanisms. In this study, mulberry G-protein genes, MaGα, MaGß, MaGγ1, and MaGγ2, were independently transformed into tobacco, and the transgenic plants were used for resistance identification experiments. The ectopic expression of MaGα in tobacco decreased the tolerance to drought and salt stresses, while the overexpression of MaGß, MaGγ1, and MaGγ2 increased the tolerance. Further analysis showed that mulberry G-proteins may regulate drought and salt tolerances by modulating reactive oxygen species' detoxification. This study revealed the roles of each mulberry G-protein subunit in abiotic stress tolerance and advances our knowledge of the molecular mechanisms underlying G-proteins' regulation of plant abiotic stress tolerance.

Plant G-protein ß subunits positively regulate drought tolerance by elevating detoxification of ROS.

Heterotrimeric guanine-nucleotide-binding proteins (G-proteins) consist of α, ß and γ subunits and play important roles in response and tolerance to abiotic stresses in plants, but the function of the heterotrimeric G-protein ß subunit in response to drought remains unclear. In the present study, the AGB1 mutants (agb1-2-1 and agb1-3-2) were more sensitive to drought than the wild-type. The overexpression of mulberry (Morus alba L.) G-protein ß subunit in transgenic tobacco (Nicotiana tabacum L.) significantly enhanced the plants' drought tolerance. The transgenic tobacco plants had higher proline contents and peroxidase activities, and lower malonaldehyde and hydrogen peroxide contents and superoxide free radical accumulations under drought conditions. Additionally, transcript levels of the tobacco antioxidative genes, NtSOD and NtCAT, increased in drought-stressed transgenic tobacco plants. Thus, the heterotrimeric G-protein ß subunits positively regulate drought tolerance in plants.

[Biological and epidemiological characteristics of the pathogen of hypertrophy sorosis scleroteniosis, Ciboria shiraiana].

Objective: We studied the biological and the epidemiological characteristics of the pathogen of hypertrophy sorosis scleroteniosis, which is a devastating fungal disease of mulberry. Methods: We studied the asexual and sexual reproductive phase stages of C. shiraiana, including the infection ability of hyphal, dormancy of sclerotia, the structures, release, number and germination of ascospores from apothecia, as well as the phenology of sclerotial germination. Results: In C. shiraiana, hyphae had no infection ability toward the female flowers of mulberry. Sclerotia of C. shiraiana must undergo cold treatment above 6 weeks, then the dormancy-breaking sclerotia could germinate to apothecia. One to fifteen apothecia were germinated from one sclerotium, and the number of ascospores in a 1.5 cm diameter apothecia could contain up to (5.6-6.3)×107. Ascospore C. shiraiana had significantly higher germination rates in acid than in neutral and alkaline environments. From late January to middle April, sclerotia germinated to apothecia, and got the highest value in the middle of March. Conclusion: C. shiraiana is a formidable pathogen to cause epidemic disease and damage in mulberry.

Highly efficient and inducible DNA excision in transgenic silkworms using the FLP/FRT site-specific recombination system.

Efficient and inducible recombinase-mediated DNA excision is an optimal technology for automatically deleting unwanted DNA sequences, including selection marker genes. However, this methodology has yet to be established in transgenic silkworms. To achieve efficient and inducible FLP recombinase-mediated DNA excision in transgenic silkworms, one transgenic target strain (TTS) containing an FRT-flanked silkworm cytoplasmic actin 3 gene promoter (A3)-enhanced green fluorescent protein (EGFP) expression cassette, as well as two different types of FLP recombinase expression helper strains were generated. Then, the FLP recombinase was introduced into the TTS silkworms by pre-blastoderm microinjection and sexual hybridization. Successful recombinase-mediated deletion of the A3-EGFP expression cassette was observed in the offspring of the TTS, and the excision efficiencies of the FLP expression vector and FLP mRNA pre-blastoderm microinjection were 2.38 and 13.3 %, respectively. The excision efficiencies resulting from hybridization between the TTS and the helper strain that contained a heat shock protein 70 (Hsp70)-FLP expression cassette ranged from 32.14 to 36.67 % after heat shock treatment, while the excision efficiencies resulting from hybridization between the TTS and the helper strain containing the A3-FLP expression cassette ranged from 97.01 to 100 %. These results demonstrate that the FLP/FRT system can be used to achieve highly efficient and inducible post-integration excision of unwanted DNA sequences in transgenic silkworms in vivo. Our present study will facilitate the development and application of the FLP/FRT system for the functional analysis of unknown genes, and establish the safety of transgenic technologies in the silkworm and other lepidopteran species.

[Introduction trial of medicine mulberry (Morus nigra) in Chongqing].

Medicine mulberry (Morus nigra) mainly distributed in southern areas of Xinjiang Uighur Autonomous Region and introduced by grafting, is a unique Morus species, whose plant number is little. As a traditional herbal medicine, medicine mulberry with high levels of secondary metabolites has important values of scientific research and utilization. In order to solve the introduction problems for medicine mulberry, we have established its rapid propagation system through tissue culture since 2011. The shoots of medicine mulberry through tissue culture were transplanted into the field to carry out an introduction experiment. Here, we firstly reported that the growth status and pest and disease occurrence of medicine mulberry in the field of Chongqing and found that the medicine mulberry through tissue culture had well-developed root system, it showed better growth than medicine mulberry by grafting technique, and Pseudodendrothrips moil was a major pest of medicine mulberry. The introduction technique for medicine mulberry established successfully in this study could lay the foundation for large-scale cultivation and high efficiency utilization of medicine mulberry.

Safe utilization and remediation potential of the mulberry-silkworm system in heavy metal-contaminated lands: A review.

Mulberry cultivation and silkworm rearing hold a prominent position in the agricultural industries of many Asian countries, contributing to economic growth, sustainable development, and cultural heritage preservation. Applying the soil-mulberry-silkworm system (SMSS) to heavy metal (HM)-contaminated areas is significant economically, environmentally, and socially. The ultimate goal of this paper is to review the main research progress of SMSS under HM stress, examining factors affecting its safe utilization and remediation potential for HM-contaminated soils. HM tolerance of mulberry and silkworms relates to their growth stages. Based on the standards for HM contaminants in various mulberry and silkworm products and the bioconcentration factor of HMs at different parts of SMSS, we calculated maximum safe Cd and Pb levels for SMSS application on contaminated lands. Several remediation practices demonstrated mulberry's ability to grow on barren lands, absorb various HMs, while silkworm excreta can adsorb HMs and improve soil fertility. Considering multiple factors influencing HM tolerance and accumulation, we propose a decision model to guide SMSS application in polluted areas. Finally, we discussed the potential of using molecular breeding techniques to screen or develop varieties better suited for HM-contaminated regions. However, actual pollution scenarios are often complex, requiring consideration of multiple factors. More large-scale applications are crucial to enhance the theoretical foundation for applying SMSS in HM pollution risk areas.

An efficient and safe strategy for germ cell-specific automatic excision of foreign DNA in F1 hybrid transgenic silkworms.

The safety of transgenic technology is a major obstacle in the popularization and use of transgenic silkworms and their products. In sericulture, only the first filial generation (F1 ) hybrid eggs produced by cross-breeding Japanese and Chinese original strains are usually used for the large-scale breeding of silkworms, but this may result in uncontrolled transgene dispersal during the popularization and application of the F1 hybrid transgenic eggs. To address this issue, we developed a safe and efficient strategy using the GAL4/Upstream activating sequence (UAS) system, the FLP/flippase recognition target (FRT) system, and the gonad-specific expression gene promoters (RSHP1p and Nanosp) for the germ cell-specific automatic excision of foreign DNA in the F1 hybrid transgenic silkworms. We established 2 types of activator strains, R1p::GAL4-Gr and Nsp::GAL4-Gr, containing the testis-specific GAL4 gene expression cassettes driven by RSHP1p or Nanosp, respectively, and 1 type of effector strain, UAS::FLP-Rg, containing the UAS-linked FLP gene expression cassette. The FLP recombinase-mediated sperm-specific complete excision of FRT-flanked target DNA in the F1 double-transgenic silkworms resulting from the hybridization of R1p::GAL4-Gr and UAS::FLP-Rg was 100%, whereas the complete excision efficiency resulting from the hybridization of Nsp::GAL4-Gr and UAS::FLP-Rg ranged from 13.73% to 80.3%. Additionally, we identified a gene, sw11114, that is expressed in both testis and ovary of Bombyx mori, and can be used to establish novel gonad-specific expression systems in transgenic silkworms. This strategy has the potential to fundamentally solve the safety issue in the production of F1 transgenic silkworm eggs and provides an important reference for the safety of transgenic technology in other insect species.

Haplotype-resolved chromosomal-level genome assembly reveals regulatory variations in mulberry fruit anthocyanin content.

Understanding the intricate regulatory mechanisms underlying the anthocyanin content (AC) in fruits and vegetables is crucial for advanced biotechnological customization. In this study, we generated high-quality haplotype-resolved genome assemblies for two mulberry cultivars: the high-AC 'Zhongsang5801' (ZS5801) and the low-AC 'Zhenzhubai' (ZZB). Additionally, we conducted a comprehensive analysis of genes associated with AC production. Through genome-wide association studies (GWAS) on 112 mulberry fruits, we identified MaVHAG3, which encodes a vacuolar-type H+-ATPase G3 subunit, as a key gene linked to purple pigmentation. To gain deeper insights into the genetic and molecular processes underlying high AC, we compared the genomes of ZS5801 and ZZB, along with fruit transcriptome data across five developmental stages, and quantified the accumulation of metabolic substances. Compared to ZZB, ZS5801 exhibited significantly more differentially expressed genes (DEGs) related to anthocyanin metabolism and higher levels of anthocyanins and flavonoids. Comparative analyses revealed expansions and contractions in the flavonol synthase (FLS) and dihydroflavonol 4-reductase (DFR) genes, resulting in altered carbon flow. Co-expression analysis demonstrated that ZS5801 displayed more significant alterations in genes involved in late-stage AC regulation compared to ZZB, particularly during the phase stage. In summary, our findings provide valuable insights into the regulation of mulberry fruit AC, offering genetic resources to enhance cultivars with higher AC traits.

A mulberry 9-cis-epoxycarotenoid dioxygenase gene MaNCED1 is involved in plant growth regulation and confers salt and drought tolerance in transgenic tobacco.

The phytohormone abscisic acid (ABA) is vital in regulating root elongation, seed germination, and abiotic stress responses in plants. Conversely, the mechanisms of ABA in mulberry root growth, seed germination, and abiotic stress responses are poorly understood. Here, we reported that exogenous ABA and drought treatment inhibited the growth of mulberry seedlings but significantly increased the ratio of root/stem. Inhibition of ABA synthesis by fluridone and sodium tungstate resulted in the decrease of root/stem ratio. We also showed that the expression of MaNCED1 in the root was strongly induced by drought and salt stress. Increasing the expression of MaNCED1 in tobacco using overexpression leads to increased root elongation and reduced seed germination. Compared with the wild type, the accumulation of H2O2 and MDA was reduced, while the POD activity and proline content was increased in the transgenic plants after drought and salt treatment. Further studies revealed increased resistance to drought and salt stress in MaNCED1 overexpressed tobaccos. Meanwhile, the auxin and ethylene signal pathway-related gene expression levels increased in MaNCED1 overexpressed tobaccos. This study demonstrated the roles of mulberry MaNCED1 in regulating plant development and abiotic stress responses. It gave further insights into the coordinated regulation of ABA, auxin, and ethylene in seed growth and germination.

Stigma type and transcriptome analyses of mulberry revealed the key factors associated with Ciboria shiraiana resistance.

Ciboria shiraiana is a fungal pathogen and the causal agent of hypertrophy sorosis scleroteniosis (HSS) in mulberry, leading to substantial economic losses in the mulberry fruit-related industry. To obtain HSS resistant resources and investigate the resistance mechanism, the resistances of 14 mulberry varieties were assessed. Morus laevigata Wall. (MLW) varieties showed strong resistance to C. shiraiana, and the pathogen's infection was associated with mulberry fluorescence. Stigmas were identified as the infection site through cutting experiments. Susceptible varieties (S-varieties) displayed secretory droplets on their stigma papillar cell surfaces, while MLWs lacked these secretions. Correlation analysis between the secretion rate and the diseased fruit rate indicated that the differences between resistant varieties (R-varieties) and S-varieties were related to the stigma type. Furthermore, comparative transcriptome analysis was performed on stigma and ovary samples from R- and S-varieties. Compared with the stigma of R-varieties, the key differentially expressed genes (DEGs) with significantly higher expression in S-variety stigmas mainly participated in the fatty acid biosynthetic process. In R-variety stigmas and ovaries, the transcript levels of DEGs involved in defense response, including resistance (R) genes, were significantly higher than that of S-varieties. Overexpression of MlwRPM1-2 and MlwRGA3 enhances resistance to C. shiraiana and Sclerotinia sclerotiorum, but not Botrytis cinerea in tobacco. These findings help us explain the different resistance mechanisms of mulberry to C. shiraiana, and the critical defense genes in R-varieties can be applied to breeding antifungal plant varieties.

[Progress in Cre/lox site-specific recombination system in higher eukaryotes].

Cre/lox system derived from P1 bacteriaphage can quickly and effectively achieve gene insertion, deletion, replacement, and inversion by means of site-specific recombination. As one of the most important tools for gene targeting at present, Cre/lox system has been widely used in Arabidopsis thaliana, Oryza sativa L., Mus musculus, Drosophila melanogaster, Danio rerio, and other higher eukaryotic organisms. This review roundly described the basic profile of Cre/lox system, and its application in higher eukaryotes. In addition, we also discussed the main problems and developmental trend of the Cre/lox system in this review, which can be a good reference for using Cre/lox system to realize the gene manipulations of the different high eukaryotic organisms.

Ectopic overexpression of mulberry MnT5H2 enhances melatonin production and salt tolerance in tobacco.

Soil salinization severely inhibits plant growth and has become one of the major limiting factors for global agricultural production. Melatonin (N-acetyl-5-methoxytryptamine) plays an important role in regulating plant growth and development and in responding to abiotic stresses. Tryptamine-5-hydroxylase (T5H) is an enzyme essential for the biosynthesis of melatonin in plants. Previous studies have identified the gene MnT5H for melatonin synthesis in mulberry (Morus notabilis), but the role of this gene in response to salinity stress in mulberry is remain unclear. In this study, we ectopically overexpressed MnT5H2 in tobacco (Nicotiana tabacum L.) and treated it with NaCl solutions. Compared to wild-type (WT), melatonin content was significantly increased in the overexpression-MnT5H2 tobacco. Under salt stress, the expression of NtCAT, NtSOD, and NtERD10C and activity of catalase (CAT), peroxidase (POD), and the content of proline (Pro) in the transgenic lines were significantly higher than that in WT. The Malondialdehyde (MDA) content in transgenic tobacco was significantly lower than that of WT. Furthermore, transgenic tobacco seedlings exhibited faster growth in media with NaCl. This study reveals the changes of melatonin and related substance content in MnT5H2-overexpressing tobacco ultimately lead to improve the salt tolerance of transgenic tobacco, and also provides a new target gene for breeding plant resistance to salt.

Very Early Corona Treatment-Mediated Artificial Incubation of Silkworm Eggs and Germline Transformation of Diapause Silkworm Strains.

Diapause is an important biological characteristic for many insect species to adapt to adverse environmental conditions and maintain the continuity of the race. Compared with the traditional hydrochloric acid or/and cold storage treatment methods, the artificial corona incubation technology of silkworm (Bombyx mori) eggs has many advantages including, the absence of pollution, easy operation and safety. However, this technology has not yet been applied in sericulture. In this study, we developed a novel artificial corona instrument to successfully disrupt the diapause of newly laid and refrigerated eggs from various Chinese and Japanese lineage silkworm strains. Subsequently, we invented a very early corona treatment (VECT) strategy to prevent the diapause of newly laid silkworm eggs within 4 h of oviposition. The hatching rates of the larvae were more than 95% in all diapause silkworm strains, which was comparable to the effect of the traditional HCl treatment strategy. In addition, we developed a combination strategy of VECT and pre-blastoderm microinjection and successfully created transgenic silkworms in various diapause strains. The results of the current study can aid in improving the corona artificial incubation technology and promote its application in sericulture.

Chromosome-level Genomes Reveal the Genetic Basis of Descending Dysploidy and Sex Determination in Morus Plants.

Multiple plant lineages have independently evolved sex chromosomes and variable karyotypes to maintain their sessile lifestyles through constant biological innovation. Morus notabilis, a dioecious mulberry species, has the fewest chromosomes among Morus spp., but the genetic basis of sex determination and karyotype evolution in this species has not been identified. In this study, three high-quality genome assemblies were generated for Morus spp. [including dioecious M. notabilis (male and female) and Morus yunnanensis (female)] with genome sizes of 301-329 Mb and were grouped into six pseudochromosomes. Using a combination of genomic approaches, we found that the putative ancestral karyotype of Morus species was close to 14 protochromosomes, and that several chromosome fusion events resulted in descending dysploidy (2n = 2x = 12). We also characterized a ∼ 6.2-Mb sex-determining region on chromosome 3. Four potential male-specific genes, a partially duplicatedDNA helicase gene (named MSDH) and three Ty3_Gypsy long terminal repeat retrotransposons (named MSTG1/2/3), were identified in the Y-linked area and considered to be strong candidate genes for sex determination or differentiation. Population genomic analysis showed that Guangdong accessions in China were genetically similar to Japanese accessions of mulberry. In addition, genomic areas containing selective sweeps that distinguish domesticated mulberry from wild populations in terms of flowering and disease resistance were identified. Our study provides an important genetic resource for sex identification research and molecular breeding in mulberry.

An APSES Transcription Factor Xbp1 Is Required for Sclerotial Development, Appressoria Formation, and Pathogenicity in Ciboria shiraiana.

Sclerotinia diseases are important plant fungal diseases that, causes huge economic worldwide losses every year. Ciboria shiraiana is the main pathogen that results in mulberry sclerotia diseases. Sclerotia and appressoria play important roles in long-term pathogen survival and in host infection during life and disease cycles. However, the molecular mechanisms of sclerotial development and appressoria formation in C. shiraiana have not been well studied. Here, an Asm1p, Phd1p, Sok2p, Efg1p and StuAp (APSES)-type transcription factor in C. shiraiana, CsXbp1, involved in sclerotial development and appressoria formation was functionally characterized. Bioinformatics analyses showed that CsXbp1 contained an APSES-type DNA binding domain. The expression levels of CsXbp1 were higher in sclerotia and during later stages of infection. Compared with wild-type strains, hyphal growth was slower, the number and weight of sclerotia were reduced significantly, and appressoria formation was obviously delayed in CsXbp1 RNA interference (RNAi) strains. Moreover, the CsXbp1 RNAi strains showed weakened pathogenicity owing to compound appressoria defects. Tobacco rattle virus-mediated host-induced gene silencing enabled Nicotiana benthamiana to increase its resistance to C. shiraiana by reducing the CsXbp1 transcripts level. Thus, CsXbp1 plays vital roles in sclerotial formation, appressoria formation, and pathogenicity in C. shiraiana. This study provides new insights into the infection mechanisms of C. shiraiana and plant resistance breeding.

The C-terminal tail of the plant endosomal-type NHXs plays a key role in its function and stability.

Typically, Na+/H+ antiporters (NHXs) possess a conserved N-terminus for cation binding and exchange and a hydrophilic C-terminus for regulating the antiporter activity. Plant endosomal-type NHXs play important roles in protein trafficking, as well as K+ and vesicle pH homeostasis, however the role of the C-terminal tail remains unclear. Here, the function of MnNHX6, an endosomal-type NHX in mulberry, was investigated using heterologous expression in yeast. Functional and localization analyses of C-terminal truncation and mutations in MnNHX6 revealed that the C-terminal conserved region was responsible for the function and stability of the protein and its hydrophobicity, which is a key domain requirement. Nuclear magnetic resonance spectroscopy provided direct structural evidence and yeast two-hybrid screening indicated that this functional domain was also necessary for interaction with sorting nexin 1. Our findings demonstrate that although the C-terminal tail of MnNHX6 is intrinsically disordered, the C-terminal conserved region may be an important part of the external mouth of this transporter, which controls protein function and stability by serving as an inter-molecular cork with a chain mechanism. These findings improve our understanding of the roles of the C-terminal tail of endosomal-type NHXs in plants and the ion transport mechanism of NHX-like antiporters.