Whitefly hijacks a plant detoxification gene that neutralizes plant toxins.

Plants protect themselves with a vast array of toxic secondary metabolites, yet most plants serve as food for insects. The evolutionary processes that allow herbivorous insects to resist plant defenses remain largely unknown. The whitefly Bemisia tabaci is a cosmopolitan, highly polyphagous agricultural pest that vectors several serious plant pathogenic viruses and is an excellent model to probe the molecular mechanisms involved in overcoming plant defenses. Here, we show that, through an exceptional horizontal gene transfer event, the whitefly has acquired the plant-derived phenolic glucoside malonyltransferase gene BtPMaT1. This gene enables whiteflies to neutralize phenolic glucosides. This was confirmed by genetically transforming tomato plants to produce small interfering RNAs that silence BtPMaT1, thus impairing the whiteflies' detoxification ability. These findings reveal an evolutionary scenario whereby herbivores harness the genetic toolkit of their host plants to develop resistance to plant defenses and how this can be exploited for crop protection.

Small peptide signaling via OsCIF1/2 mediates Casparian strip formation at the root endodermal and nonendodermal cell layers in rice.

The Casparian strip (CS) is a ring-like lignin structure deposited between endodermal cells that forms an apoplastic barrier to control the selective uptake of nutrients in vascular plants. However, the molecular mechanism of CS formation in rice (Oryza sativa), which possesses one CS each in the endodermis and exodermis, is relatively unknown. Here, we functionally characterized CS INTEGRITY FACTOR1 (OsCIF1a, OsCIF1b), OsCIF2, and SCHENGEN3 (OsSGN3a, OsSGN3b) in rice. OsCIF1s and OsCIF2 were mainly expressed in the stele, while OsSGN3s localized around the CS at the endodermis. Knockout of all three OsCIFs or both OsSGN3s resulted in a discontinuous CS and a dramatic reduction in compensatory (less localized) lignification and suberization at the endodermis. By contrast, ectopic overexpression of OsCIF1 or OsCIF2 induced CS formation as well as overlignification and oversuberization at single or double cortical cell layers adjacent to the endodermis. Ectopic co-overexpression of OsCIF1 and SHORTROOT1 (OsSHR1) induced the formation of more CS-like structures at multiple cortical cell layers. Transcriptome analysis identified 112 downstream genes modulated by the OsCIF1/2-OsSGN3 signaling pathway, which is involved in CS formation and activation of the compensatory machinery in native endodermis and nonnative endodermis-like cell layers. Our results provide important insights into the molecular mechanism of CIF-mediated CS formation at the root endodermal and nonendodermal cell layers.

Regulation by distinct MYB transcription factors defines the roles of OsCYP86A9 in anther development and root suberin deposition.

Cytochrome P450 proteins (CYPs) play critical roles in plant development and adaptation to fluctuating environments. Previous reports have shown that CYP86A proteins are involved in the biosynthesis of suberin and cutin in Arabidopsis. However, the functions of these proteins in rice remain obscure. In this study, a rice mutant with incomplete male sterility was identified. Cytological analyses revealed that this mutant was defective in anther development. Cloning of the mutant gene indicated that the responsible mutation was on OsCYP86A9. OsMYB80 is a core transcription factor in the regulation of rice anther development. The expression of OsCYP86A9 was abolished in the anther of osmyb80 mutant. In vivo and in vitro experiments showed that OsMYB80 binds to the MYB-binding motifs in OsCYP86A9 promoter region and regulates its expression. Furthermore, the oscyp86a9 mutant exhibited an impaired suberin deposition in the root, and was more susceptible to drought stress. Interestingly, genetic and biochemical analyses revealed that OsCYP86A9 expression was regulated in the root by certain MYB transcription factors other than OsMYB80. Moreover, mutations in the MYB genes that regulate OsCYP86A9 expression in the root did not impair the male fertility of the plant. Taken together, these findings revealed the critical roles of OsCYP86A9 in plant development and proposed that OsCYP86A9 functions in anther development and root suberin formation via two distinct tissue-specific regulatory pathways.

Three OsMYB36 members redundantly regulate Casparian strip formation at the root endodermis.

Plants have evolved a lignin-based Casparian strip (CS) in roots that restricts passive diffusion of mineral elements from the soil to the stele. However, the molecular mechanisms underlying CS formation in rice (Oryza sativa), which contains a CS at both the exodermis and endodermis, are poorly understood. Here, we demonstrate that CS formation at the rice endodermis is redundantly regulated by three MYELOBLASTOSIS (MYB) transcription factors, OsMYB36a, OsMYB36b, and OsMYB36c, that are highly expressed in root tips. Knockout of all three genes resulted in a complete absence of CS at the endodermis and retarded plant growth in hydroponic conditions and in soil. Compared with the wild-type, the triple mutants showed higher calcium (Ca) levels and lower Mn, Fe, Zn, Cu, and Cd levels in shoots. High Ca supply further inhibited mutant growth and increased Ca levels in shoots. Transcriptome analysis identified 1,093 downstream genes regulated by OsMYB36a/b/c, including the key CS formation gene OsCASP1 and other genes that function in CS formation at the endodermis. Three OsMYB36s regulate OsCASP1 and OsESB1 expression by directly binding to MYB-binding motifs in their promoters. Our findings thus provide important insights into the mechanism of CS formation at the endodermis and the selective uptake of mineral elements in roots.

The transcription factor OsbZIP48 governs rice responses to zinc deficiency.

Zinc (Zn) deficiency is the most prevalent micronutrient disorder in rice and leads to delayed development and decreased yield. Nevertheless, despite its primary importance, how rice responds to Zn deficiency remains poorly understood. This study presents genetic evidence supporting the crucial role of OsbZIP48 in regulating rice's response to Zn deficiency, consistent with earlier findings in the model plant Arabidopsis. Genetic inactivation of OsbZIP48 in rice seedlings resulted in heightened sensitivity to Zn deficiency and reduced Zn translocation from roots to shoots. Consistently, OsbZIP48 was constitutively expressed in roots, slightly induced by Zn deficiency in shoots and localized into nuclei induced by Zn deficiency. Comparative transcriptome analysis of the wild-type plants and osbzip48 mutant grown under Zn deficiency enabled the identification of OsbZIP48 target genes, including key Zn transporter genes (OsZIP4 and OsZIP8). We demonstrated that OsbZIP48 controlled the expressions of these genes by directly binding to their promoters, specifically to the Zn deficiency response element motif. This study establishes OsbZIP48 as a critical transcription factor in rice's response to Zn deficiency, offering valuable insights for developing Zn-biofortified rice varieties to combat global Zn limitation.

MAPK-directed activation of the whitefly transcription factor CREB leads to P450-mediated imidacloprid resistance.

The evolution of insect resistance to pesticides poses a continuing threat to agriculture and human health. While much is known about the proximate molecular and biochemical mechanisms that confer resistance, far less is known about the regulation of the specific genes/gene families involved, particularly by trans-acting factors such as signal-regulated transcription factors. Here we resolve in fine detail the trans-regulation of CYP6CM1, a cytochrome P450 that confers resistance to neonicotinoid insecticides in the whitefly Bemisia tabaci, by the mitogen-activated protein kinase (MAPK)-directed activation of the transcription factor cAMP-response element binding protein (CREB). Reporter gene assays were used to identify the putative promoter of CYP6CM1, but no consistent polymorphisms were observed in the promoter of a resistant strain of B. tabaci (imidacloprid-resistant, IMR), which overexpresses this gene, compared to a susceptible strain (imidacloprid-susceptible, IMS). Investigation of potential trans-acting factors using in vitro and in vivo assays demonstrated that the bZIP transcription factor CREB directly regulates CYP6CM1 expression by binding to a cAMP-response element (CRE)-like site in the promoter of this gene. CREB is overexpressed in the IMR strain, and inhibitor, luciferase, and RNA interference assays revealed that a signaling pathway of MAPKs mediates the activation of CREB, and thus the increased expression of CYP6CM1, by phosphorylation-mediated signal transduction. Collectively, these results provide mechanistic insights into the regulation of xenobiotic responses in insects and implicate both the MAPK-signaling pathway and a transcription factor in the development of pesticide resistance.

Overexpression of an ART1-Interacting Gene OsNAC016 Improves Al Tolerance in Rice.

Rice (Oryza sativa) exhibits tremendous aluminum (Al)-tolerance. The C2H2-transcription factor (TF) ART1 critically regulates rice Al tolerance via modulation of specific gene expression. However, little is known about the posttranscriptional ART1 regulation. Here, we identified an ART1-interacted gene OsNAC016 via a yeast two-hybrid (Y2H) assay. OsNAC016 was primarily expressed in roots and weakly induced by Al. Immunostaining showed that OsNAC016 was a nuclear protein and localized in all root cells. Knockout of OsNAC016 did not alter Al sensitivity. Overexpression of OsNAC016 resulted in less Al aggregation within roots and enhanced Al tolerance in rice. Based on transcriptomic and qRT-PCR evaluations, certain cell-wall-related or ART-regulated gene expressions such as OsMYB30 and OsFRDL4 were altered in OsNAC016-overexpressing plants. These results indicated that OsNAC016 interacts with ART1 to cooperatively regulate some Al-tolerance genes and is a critical regulatory factor in rice Al tolerance.

OsCASP1 Is Required for Casparian Strip Formation at Endodermal Cells of Rice Roots for Selective Uptake of Mineral Elements.

In response to diverse environmental conditions, rice (Oryza sativa) roots have developed one Casparian strip (CS) at the exodermis and one CS at the endodermis. Here, we functionally characterized OsCASP1 (Casparian strip domain protein 1) in rice. OsCASP1 was mainly expressed in the root elongation zone, and the protein encoded was first localized to all sides of the plasma membrane of endodermal cells without CS, followed by the middle of the anticlinal side of endodermal cells with CS. Knockout of OsCASP1 resulted in a defect of CS formation at the endodermis and decreased growth under both soil and hydroponic conditions. Mineral analysis showed that the oscasp1 mutants accumulated more Ca, but less Mn, Zn, Fe, Cd, and As in the shoots compared with the wild type. The growth inhibition of the mutants was further aggravated by high Ca in growth medium. The polar localization of the Si transporter Low Si 1 at the distal side of the endodermis was not altered in the mutant, but the protein abundance was decreased, resulting in a substantial reduction in silicon uptake. These results indicated that OsCASP1 is required for CS formation at the endodermis and that the CS in rice plays an important role in root selective uptake of mineral elements, especially Ca and Si.

The WRKY Transcription Factor OsWRKY54 Is Involved in Salt Tolerance in Rice.

Salt stress is a critical limiting factor for rice growth and production. Although numerous salt-tolerant genes have been identified, the mechanism underlying salt stress tolerance in rice remains unclear. This study reports the need for an uncharacterized WRKY transcription factor OsWRKY54 for rice salt-tolerance. Salt stress resulted in a rapid induction of OsWRKY54 expression in roots. Immunostaining analysis showed that it was mainly expressed in the stele. The loss of OsWRKY54 resulted in greater Na accumulation in shoots and enhanced sensitivity of rice plants to salt stress. The real-time quantitative PCR (qRT-PCR) and transcriptome analysis revealed that OsWRKY54 regulated the expression of some essential genes related to salt tolerance, such as OsNHX4 and OsHKT1;5. Furthermore, OsWRKY54 was found to regulate OsHKT1;5 expression by directly binding to the W-box motif in its promoter. Thus, these results indicated that OsWRKY54 was a critical regulatory factor in salt tolerance in rice.

Dysfunction of the 4-coumarate:coenzyme A ligase 4CL4 impacts aluminum resistance and lignin accumulation in rice.

The root cell wall is the first and primary target of aluminum (Al) toxicity. Monocots such as rice (Oryza sativa) can accumulate appreciable levels of hydroxycinnamic acids (HCAs) to modify and cross-link hemicellulose and/or lignin of the cell wall. Nevertheless, it is unclear whether this HCA-mediated modification of the cell wall is important for Al accumulation and resistance. We previously isolated and characterized a rice ral1 (resistance to aluminum 1) mutant that shows enhanced Al resistance. In this study, we cloned RAL1 and found that it encodes the 4-coumarate:coenzyme A ligase 4CL4, an enzyme putatively involved in lignin biosynthesis. Mutation of RAL1/4CL4 reduces lignin content and increases the accumulation of its substrates 4-coumaric acid (PA) and ferulic acid (FA). We demonstrate that altered lignin accumulation is not required for the enhanced Al resistance in ral1/4cl4 mutants. We found that the increased accumulation of PA and FA can reduce Al binding to hemicellulose and consequently enhance Al resistance in ral1/4cl4 mutants. Al stress is able to trigger PA and FA accumulation, which is likely caused by the repression of the expression of RAL1/4CL4 and its homologous genes. Our results thus reveal that Al-induced PA and FA accumulation is actively and positively involved in Al resistance in rice through the modification of the cell wall and thereby the reduced Al binding to the cell wall.

A NAC transcription factor OsNAC3 positively regulates ABA response and salt tolerance in rice.

BACKGROUND: NAC (NAM, ATAF and CUC) transcription factors (TFs) play vital roles in plant development and abiotic stress tolerance. Salt stress is one of the most limiting factors for rice growth and production. However, the mechanism underlying salt tolerance in rice is still poorly understood. RESULTS: In this study, we functionally characterized a rice NAC TF OsNAC3 for its involvement in ABA response and salt tolerance. ABA and NaCl treatment induced OsNAC3 expression in roots. Immunostaining showed that OsNAC3 was localized in all root cells. OsNAC3 knockout decreased rice plants' sensitivity to ABA but increased salt stress sensitivity, while OsNAC3 overexpression showed an opposite effect. Loss of OsNAC3 also induced Na+ accumulation in the shoots. Furthermore, qRT-PCR and transcriptomic analysis were performed to identify the key OsNAC3 regulated genes related to ABA response and salt tolerance, such as OsHKT1;4, OsHKT1;5, OsLEA3-1, OsPM-1, OsPP2C68, and OsRAB-21. CONCLUSIONS: This study shows that rice OsNAC3 is an important regulatory factor in ABA signal response and salt tolerance.

Comparative transcriptome analysis of differentially expressed genes in Bradysia odoriphaga Yang et Zhang (Diptera: Sciaridae) at different acute stress temperatures.

The gnat, Bradysia odoriphaga Yang et Zhang, is an important underground pest in Asia. B. odoriphaga differ in heat and cold tolerance and exhibit quite different developmental strategies. To understand the underlying mechanisms, we sequenced and compared the transcriptome of B. odoriphaga under 40 °C (a stressful high temperature), 25 °C, and 4 °C (a stressful low temperature) for 1 h. We found that metabolism- and ribosome-related genes were modulated. In high temperature (40 °C), heat shock protein (HSP) genes, detoxication genes, metabolism genes, protein turnover genes, and stress signal transduction genes were differentially expressed. In low temperature (4 °C), genes related with heat shock protein (HSP) and detoxication were differentially expressed. Our study increases our understanding of the complex molecular mechanisms involved in the responses of B. odoriphaga to acute temperature stress and provides a potential strategy for pest management.

Overexpression of OsCASP1 Improves Calcium Tolerance in Rice.

The Casparian strip domain protein 1 (OsCASP1) is necessary for the formation of the Casparian strip (CS) in the rice endodermis. It also controls Ca2+ transport to the stele. Here, we demonstrated that OsCASP1 overexpression enhanced Ca tolerance in rice. Under normal conditions, OsCASP1-overexpressed lines showed similar concentrations of essential metals in the roots and shoots compared to the wild type, while under high Ca conditions, Ca in the roots, shoots, and xylem sap of the OsCASP1-overexpressed lines was significantly decreased. This did not apply to other essential metals. Ca-inhibited growth was significantly alleviated in the OsCASP1-overexpressed lines. Furthermore, OsCASP1 overexpression resulted in earlier formation of both the CS and functional apoplastic barrier in the endodermis but did not induce ectopic CS formation in non-endodermal cell layers and affect suberin accumulation in the endodermis. These results indicate that the overexpression of OsCASP1 promotes CS formation in endodermal cells and inhibits Ca2+ transport by the apoplastic pathway, restricting Ca accumulation in the roots and shoots under high Ca conditions. Taken together, the results suggest that OsCASP1 overexpression is an effective way to improve rice adaptation to high Ca environments.

OsC2DP, a Novel C2 Domain-Containing Protein Is Required for Salt Tolerance in Rice.

Salt stress is one of the major factors limiting crop production globally, including rice (Oryza sativa). Although a number of genes involved in salt tolerance have been functionally identified, the mechanism underlying salt tolerance in rice is still poorly understood. Here, we reported a novel C2 domain-containing protein, OsC2DP required for salt tolerance in rice. OsC2DP was predominately expressed in the roots and its expression was repressed by salt stress. Transient expression of OsC2DP in rice protoplast cells showed that it was localized in the cytosol. Immunostaining further showed that OsC2DP was able to translocate from the cytosol to plasma membrane under salt conditions. Knockout of OsC2DP did not affect Na+ concentration in the roots, but increased shoot Na+ concentration, resulting in a significant sensitivity of rice to salt stress. Furthermore, the quantitative Real-time PCR and transcriptomic analysis showed that the expression level of some genes related to salt tolerance were indirectly regulated by OsC2DP, especially OsSOS1 and OsNHX4. These results indicate that OsC2DP has an important role in salt tolerance and these findings provide new insights into the regulation of OsC2DP gene for rice breeding with high salt tolerance.

Disruption of an amino acid transporter LHT1 leads to growth inhibition and low yields in rice.

BACKGROUND: Research on plant amino acid transporters was mainly performed in Arabidopsis, while our understanding of them is generally scant in rice. OsLHT1 (Lysine/Histidine transporter) has been previously reported as a histidine transporter in yeast, but its substrate profile and function in planta are unclear. The aims of this study are to analyze the substrate selectivity of OsLHT1 and influence of its disruption on rice growth and fecundity. RESULTS: Substrate selectivity of OsLHT1 was analyzed in Xenopus oocytes using the two-electrode voltage clamp technique. The results showed that OsLHT1 could transport a broad spectrum of amino acids, including basic, neutral and acidic amino acids, and exhibited a preference for neutral and acidic amino acids. Two oslht1 mutants were generated using CRISPR/Cas9 genome-editing technology, and the loss-of-function of OsLHT1 inhibited rice root and shoot growth, thereby markedly reducing grain yields. QRT-PCR analysis indicated that OsLHT1 was expressed in various rice organs, including root, stem, flag leaf, flag leaf sheath and young panicle. Transient expression in rice protoplast suggested OsLHT1 was localized to the plasma membrane, which is consistent with its function as an amino acid transporter. CONCLUSIONS: Our results indicated that OsLHT1 is an amino acid transporter with wide substrate specificity and with preference for neutral and acidic amino acids, and disruption of OsLHT1 function markedly inhibited rice growth and fecundity.

The ABC transporter ABCG36 is required for cadmium tolerance in rice.

Cadmium (Cd) is a highly toxic heavy metal in nature, which causes severe damage to plant growth. The molecular mechanisms for Cd detoxification are poorly understood. Here, we report that a G-type ATP-binding cassette transporter, OsABCG36, is involved in Cd tolerance in rice. OsABCG36 was expressed in both roots and shoots at a low level, but expression in the roots rather than the shoots was greatly up-regulated by a short exposure to Cd. A spatial expression analysis showed that Cd-induced expression of OsABCG36 was found in both the root tip and the mature root region. Transient expression of OsABCG36 in rice protoplast cells showed that it was localized to the plasma membrane. Immunostaining showed that OsABCG36 was localized in all root cells except the epidermal cells. Knockout of OsABCG36 resulted in increased Cd accumulation in root cell sap and enhanced Cd sensitivity, but did not affect tolerance to other metals including Al, Zn, Cu, and Pb. The concentration of Cd in the shoots was similar between the knockout lines and wild-type rice. Heterologous expression of OsABCG36 in yeast showed an efflux activity for Cd, but not for Zn. Taken together, our results indicate that OsABCG36 is not involved in Cd accumulation in the shoots, but is required for Cd tolerance by exporting Cd or Cd conjugates from the root cells in rice.

Molecular Mechanisms for Coping with Al Toxicity in Plants.

Aluminum (Al) toxicity is one of the major constraints to agricultural production in acid soils. Molecular mechanisms of coping with Al toxicity have now been investigated in a range of plant species. Two main mechanisms of Al tolerance in plants are Al exclusion from the roots and the ability to tolerate Al in the roots. This review focuses on the recent discovery of novel genes and mechanisms that confer Al tolerance in plants and summarizes our understanding of the physiological, genetic, and molecular basis for plant Al tolerance. We hope this review will provide a theoretical basis for the genetic improvement of Al tolerance in plants.

Genome-Wide Analysis of Carboxylesterases (COEs) in the Whitefly, Bemisia tabaci (Gennadius).

The whitefly (Bemisia tabaci), an important invasive pest that causes severe damage to crops worldwide, has developed resistance to a variety of insecticides. Carboxylesterases (COEs) are important multifunctional enzymes involved in the growth, development, and xenobiotic metabolism of insects. However, systematic studies on the COEs of B. tabaci are scarce. Here, 42 putative COEs in different functional categories were identified in the Mediterranean species of B. tabaci (B. tabaci MED) based on a genome database and neighbor-joining phylogeny. The expression patterns of the COEs were affected by the development of B. tabaci. The expression levels of six COEs were positively correlated with the concentration of imidacloprid to which B. tabaci adults were exposed. The mortality of B. tabaci MED adults fed dsBTbe5 (67.5%) and dsBTjhe2 (58.4%) was significantly higher than the adults fed dsEGFP (41.1%) when treated with imidacloprid. Our results provide a basis for functional research on COEs in B. tabaci and provide new insight into the imidacloprid resistance of B. tabaci.

Effective reduction of cadmium accumulation in rice grain by expressing OsHMA3 under the control of the OsHMA2 promoter.

Reducing cadmium (Cd) accumulation in rice grain is an important issue for human health. The aim of this study was to manipulate both expression and tissue localization of OsHMA3, a tonoplast-localized Cd transporter, in the roots by expressing it under the control of the OsHMA2 promoter, which shows high expression in different organs including roots, nodes, and shoots. In two independent transgenic lines, the expression of OsHMA3 was significantly enhanced in all organs compared with non-transgenic rice. Furthermore, OsHMA3 protein was detected in the root pericycle cells and phloem region of both the diffuse vascular bundle and the enlarged vascular bundle of the nodes. At the vegetative stage, the Cd concentration in the shoots and xylem sap of the transgenic rice was significantly decreased, but that of the whole roots and root cell sap was increased. At the reproductive stage, the concentration of Cd, but not other essential metals, in the brown rice of transgenic lines was decreased to less than one-tenth that of the non-transgenic rice. These results indicate that expression of OsHMA3 under the control of the OsHMA2 promoter can effectively reduce Cd accumulation in rice grain through sequestering more Cd into the vacuoles of various tissues.