Identification and characterization of the Remorin gene family in Saccharum and the involvement of ScREM1.5e-1/-2 in SCMV infection on sugarcane.

Introduction: Remorins (REMs) are plant-specific membrane-associated proteins that play important roles in plant-pathogen interactions and environmental adaptations. Group I REMs are extensively involved in virus infection. However, little is known about the REM gene family in sugarcane (Saccharum spp. hyrid), the most important sugar and energy crop around world. Methods: Comparative genomics were employed to analyze the REM gene family in Saccharum spontaneum. Transcriptomics or RT-qPCR were used to analyze their expression files in different development stages or tissues under different treatments. Yeast two hybrid, bimolecular fluorescence complementation and co-immunoprecipitation assays were applied to investigate the protein interaction. Results: In this study, 65 REMs were identified from Saccharum spontaneum genome and classified into six groups based on phylogenetic tree analysis. These REMs contain multiple cis-elements associated with growth, development, hormone and stress response. Expression profiling revealed that among different SsREMs with variable expression levels in different developmental stages or different tissues. A pair of alleles, ScREM1.5e-1/-2, were isolated from the sugarcane cultivar ROC22. ScREM1.5e-1/-2 were highly expressed in leaves, with the former expressed at significantly higher levels than the latter. Their expression was induced by treatment with H2O2, ABA, ethylene, brassinosteroid, SA or MeJA, and varied upon Sugarcane mosaic virus (SCMV) infection. ScREM1.5e-1 was localized to the plasma membrane (PM), while ScREM1.5e-2 was localized to the cytoplasm or nucleus. ScREM1.5e-1/-2 can self-interact and interact with each other, and interact with VPgs from SCMV, Sorghum mosaic virus, or Sugarcane streak mosaic virus. The interactions with VPgs relocated ScREM1.5e-1 from the PM to the cytoplasm. Discussion: These results reveal the origin, distribution and evolution of the REM gene family in sugarcane and may shed light on engineering sugarcane resistance against sugarcane mosaic pathogens.

Sugarcane mosaic virus employs 6K2 protein to impair ScPIP2;4 transport of H2O2 to facilitate virus infection.

Sugarcane mosaic virus (SCMV), one of the main pathogens causing sugarcane mosaic disease, is widespread in sugarcane (Saccharum spp. hybrid) planting areas and causes heavy yield losses. RESPIRATORY BURST OXIDASE HOMOLOG (RBOH) NADPH oxidases and plasma membrane intrinsic proteins (PIPs) have been associated with the response to SCMV infection. However, the underlying mechanism is barely known. In the present study, we demonstrated that SCMV infection upregulates the expression of ScRBOHs and the accumulation of hydrogen peroxide (H2O2), which inhibits SCMV replication. All eight sugarcane PIPs (ScPIPs) interacted with SCMV-encoded protein 6K2, whereby two PIP2s (ScPIP2;1 and ScPIP2;4) were verified as capable of H2O2 transport. Furthermore, we revealed that SCMV-6K2 interacts with ScPIP2;4 via transmembrane domain 5 to interfere with the oligomerization of ScPIP2;4, subsequently impairing ScPIP2;4 transport of H2O2. This study highlights a mechanism adopted by SCMV to employ 6K2 to counteract the host resistance mediated by H2O2 to facilitate virus infection and provides potential molecular targets for engineering sugarcane resistance against SCMV.

Genome-wide identification of the class III peroxidase gene family of sugarcane and its expression profiles under stresses.

Introduction: Plant-specific Class III peroxidases (PRXs) play a crucial role in lignification, cell elongation, seed germination, and biotic and abiotic stresses. Methods: The class III peroxidase gene family in sugarcane were identified by bioinformatics methods and realtime fluorescence quantitative PCR. Results: Eighty-two PRX proteins were characterized with a conserved PRX domain as members of the class III PRX gene family in R570 STP. The ShPRX family genes were divided into six groups by the phylogenetic analysis of sugarcane, Saccharum spontaneum, sorghum, rice, and Arabidopsis thaliana. The analysis of promoter cis-acting elements revealed that most ShPRX family genes contained cis-acting regulatory elements involved in ABA, MeJA, light responsiveness, anaerobic induction, and drought inducibility. An evolutionary analysis indicated that ShPRXs was formed after Poaceae and Bromeliaceae diverged, and tandem duplication events played a critical role in the expansion of ShPRX genes of sugarcane. Purifying selection maintained the function of ShPRX proteins. SsPRX genes were differentially expressed in stems and leaves at different growth stages in S. spontaneum. However, ShPRX genes were differentially expressed in the SCMV-inoculated sugarcane plants. A qRT-PCR analysis showed that SCMV, Cd, and salt could specifically induce the expression of PRX genes of sugarcane. Discussion: These results help elucidate the structure, evolution, and functions of the class III PRX gene family in sugarcane and provide ideas for the phytoremediation of Cd-contaminated soil and breeding new sugarcane varieties resistant to sugarcane mosaic disease, salt, and Cd stresses.

ScGAIL, a sugarcane N-terminal truncated DELLA-like protein, participates in gibberellin signaling in Arabidopsis.

The hormone gibberellin (GA) is crucial for internode elongation in sugarcane. DELLA proteins are critical negative regulators of the GA signaling pathway. ScGAI encodes a DELLA protein that was previously implicated in the regulation of sugarcane culm development. Here, we characterized ScGAI-like (ScGAIL) in sugarcane, which lacked the N-terminal region but was otherwise homologous to ScGAI. ScGAIL differed from ScGAI in its chromosomal location, expression patterns, and cellular localization. Although transgenic Arabidopsis overexpressing ScGAIL were insensitive to GAs, GA synthesis was affected in these plants, suggesting that ScGAIL disrupted the GA signaling pathway. After GA treatment, the expression patterns of GA-associated genes differed between ScGAIL-overexpressing and wild-type Arabidopsis, and the degradation of AtDELLA proteins in transgenic lines was significantly inhibited compared with wild-type lines. A sugarcane GID1 gene (ScGID1) encoding a putative GA receptor was isolated and interacted with ScGAIL in a GA-independent manner. Five ScGAIL-interacting proteins were verified by yeast two-hybrid assays, and only one interacted with ScGAI. Therefore, ScGAIL may inhibit plant growth by modulating the GA signaling pathway.

Selective Interaction of Sugarcane eIF4E with VPgs from Sugarcane Mosaic Pathogens.

Eukaryotic translation initiation factor 4E (eIF4E) plays a key role in the infection of potyviruses in susceptible plants by interacting with viral genome-linked protein (VPg). Sugarcane (Saccharum spp.) production is threatened by mosaic disease caused by Sugarcane mosaic virus (SCMV), Sorghum mosaic virus (SrMV), and Sugarcane streak mosaic virus (SCSMV). In this study, two eIF4Es and their isoform eIF(iso)4E and 4E-binding protein coding genes were cloned from sugarcane cultivar ROC22 and designated SceIF4Ea, SceIF4Eb, SceIF(iso)4E, and ScnCBP, respectively. Real-time quantitative PCR analysis showed different expression profiles of these four genes upon SCMV challenge. A subcellular localization assay showed that SceIF4Ea, SceIF4Eb, SceIF(iso)4E, and ScnCBP were distributed in the nucleus and cytoplasm. Yeast two-hybrid (Y2H) and bimolecular fluorescence complementation (BiFC) assays showed that SceIF4Ea/b and SceIF(iso)4E were selectively employed by different sugarcane mosaic pathogens, i.e., SCMV-VPg interacted with SceIF4Ea/b and SceIF(iso)4E, SrMV-VPg interacted with both SceIF4Eb and SceIF(iso)4E, and SCSMV-VPg interacted only with SceIF(iso)4E. Intriguingly, the BiFC assays, but not the Y2H assays, showed that ScnCBP interacted with the VPgs of SCMV, SrMV, and SCSMV. Competitive interaction assays showed that SCMV-VPg, SrMV-VPg, and SCMV-VPg did not compete with each other to interact with SceIF(iso)4E, and SceIF(iso)4E competed with SceIF4Eb to interact with SrMV-VPg but not SCMV-VPg. This study sheds light on the molecular mechanism of sugarcane mosaic pathogen infection of sugarcane plants and benefits sugarcane breeding against the sugarcane mosaic disease.

A contribution to the knowledge of cave-adapted ground beetles from Guiyang, central Guizhou Province, southwestern China (Coleoptera, Carabidae, Trechini).

A new genus and two new species of cavernicolous trechines are reported from central Guizhou Province, southwestern China. Haixiaphaenops gen. nov. is established to place a new species discovered in two limestone caves in northern Qingzhen Shi: H.jinxiaohongae sp. nov. (Dawan Dong cave and Changtu Dong and Dawan Dong caves). This new genus is allied to Zhijinaphaenops Uéno & Ran, 2002. Zhijinaphaenopszhaofeii sp. nov. is described from Zhangkou Dong cave in northern Jiuzhuang Zhen of Xifeng County. In addition, two new localities of the species Zhijinaphaenopsjingliae Deuve & Tian, 2015, and two new localities of SinaphaenopschengguangyuaniMa et al. 2020 are provided. A distribution map for all cavernicolous trechine beetles known in Guiyang is provided.

IL-17 predicts the effect of TACE combined with apatinib in hepatocellular carcinoma.

OBJECTIVE: IL-17 is considered to be a cancer-promoting gene in hepatocellular carcinoma (HCC). Here, we explored the effect of IL-17 in predicting the therapeutic effect of transcatheter arterial chemoembolization (TACE) combined with apartinib in patients with HCC in this study. METHODS: Established of IL-17 knockdown SK-Hep1 cells for studying the effects of IL-17 expression on the invasion and migration of human HCC cells in vitro by transwell assay and tumor angiogenesis in nude mouse. Immunohistochemistry was used to detect the expression of IL-17, E-cadherin, Vimentin and CD34 protein in 175 cases of human HCC tumor tissues. Kaplan-Meier was used to analyze the prognostic significance of TACE combined with apatinib treatment in HCC patients. RESULTS: n SK-Hep1 cells, IL-17 knockdown could increase E-cadherin protein expression, reduce vimentin protein expression, inhibit cell invasion and migration in vitro, and inhibit angiogenesis of tumor and decrease plasma VEGF level in nude mouse. In tumor tissues of HCC patients, IL-17 protein expression was negatively correlated with E-cadherin protein expression (r = -0.622, P < 0.001), positively correlated with Vimentin protein expression (r = 0.540, P < 0.001), and was positively correlated with MVD of HCC tumor tissues (r = 0.564, P < 0.001). Compared with adjuvant TACE alone, patients with low-expression of IL-17 treated combined with apatinib have a higher 5-year overall survival. However, additional apatinib treatment did not significantly improve 5-year overall survival in HCC patients with high IL-17 expression. CONCLUSION: IL-17 had a pivotal role in the invasion and angiogenesis of HCC and contribute to the selection of patients who may benefit from adjuvant TACE combined with apatinib.

Remorin interacting with PCaP1 impairs Turnip mosaic virus intercellular movement but is antagonised by VPg.

Group 1 Remorins (REMs) are extensively involved in virus trafficking through plasmodesmata (PD). However, their roles in Potyvirus cell-to-cell movement are not known. The plasma membrane (PM)-associated Ca2+ binding protein 1 (PCaP1) interacts with the P3N-PIPO of Turnip mosaic virus (TuMV) and is required for TuMV cell-to-cell movement, but the underlying mechanism remains elusive. The mutant plants with overexpression or knockout of REM1.2 were used to investigate its role in TuMV cell-to-cell movement. Arabidopsis thaliana complementary mutants of pcap1 were used to investigate the role of PCaP1 in TuMV cell-to-cell movement. Yeast-two-hybrid, bimolecular fluorescence complementation, co-immunoprecipitation and RT-qPCR assays were employed to investigate the underlying molecular mechanism. The results show that TuMV-P3N-PIPO recruits PCaP1 to PD and the actin filament-severing activity of PCaP1 is required for TuMV intercellular movement. REM1.2 negatively regulates the cell-to-cell movement of TuMV via competition with PCaP1 for binding actin filaments. As a counteractive response, TuMV mediates REM1.2 degradation via both 26S ubiquitin-proteasome and autophagy pathways through the interaction of VPg with REM1.2 to establish systemic infection in Arabidopsis. This work unveils the actin cytoskeleton and PM nanodomain-associated molecular events underlying the cell-to-cell movement of potyviruses.

Identification of Sugarcane Host Factors Interacting with the 6K2 Protein of the Sugarcane Mosaic Virus.

The 6K2 protein of potyviruses plays a key role in the viral infection in plants. In the present study, the coding sequence of 6K2 was cloned from Sugarcane mosaic virus (SCMV) strain FZ1 into pBT3-STE to generate the plasmid pBT3-STE-6K2, which was used as bait to screen a cDNA library prepared from sugarcane plants infected with SCMV based on the DUALmembrane system. One hundred and fifty-seven positive colonies were screened and sequenced, and the corresponding full-length genes were cloned from sugarcane cultivar ROC22. Then, 24 genes with annotations were obtained, and the deduced proteins were classified into three groups, in which eight proteins were involved in the stress response, 12 proteins were involved in transport, and four proteins were involved in photosynthesis based on their biological functions. Of the 24 proteins, 20 proteins were verified to interact with SCMV-6K2 by yeast two-hybrid assays. The possible roles of these proteins in SCMV infection on sugarcane are analyzed and discussed. This is the first report on the interaction of SCMV-6K2 with host factors from sugarcane, and will improve knowledge on the mechanism of SCMV infection in sugarcane.

Diversity of the Bacterial Microbiome in the Roots of Four Saccharum Species: S. spontaneum, S. robustum, S. barberi, and S. officinarum.

Endophytic bacteria are nearly ubiquitously present in the internal tissues of plants, and some endophytes can promote plant growth. In this study, we sampled the roots of four ancestral species of sugarcane (two genotypes per species) and two sugarcane cultivars, and used 16S rRNA and nifH gene sequencing to characterize the root endophytic bacterial communities and diazotroph diversity. A total of 7,198 operational taxonomic units (OTUs) were detected for the endophytic bacteria community. The endophytic bacterial communities exhibited significantly different α- and ß-diversities. From the 202 detected families in the sugarcane roots, a core microbiome containing 13 families was identified. The nifH gene was successfully detected in 9 of 30 samples from the four sugarcane species assayed, and 1,734 OTUs were merged for endophytic diazotrophs. In the tested samples, 43 families of endophytic diazotrophs were detected, and six families showed differences across samples. Among the 20 most abundant detected genera, 10 have been reported to be involved in nitrogen fixation in sugarcane. These findings demonstrate the diversity of the microbial communities in different sugarcane germplasms and shed light on the mechanism of biological nitrogen fixation in sugarcane.

Dissecting the Molecular Mechanism of the Subcellular Localization and Cell-to-cell Movement of the Sugarcane mosaic virus P3N-PIPO.

The coding sequence of P3N-PIPO was cloned by fusion PCR from Sugarcane mosaic virus (SCMV), a main causal agent of sugarcane (Saccharum spp. hybrid) mosaic disease. SCMV P3N-PIPO preferentially localized to the plasma membrane (PM) compared with the plasmodesmata (PD), as demonstrated by transient expression and plasmolysis assays in the leaf epidermal cells of Nicotiana benthamiana. The subcellular localization of the P3N-PIPO mutants P3N-PIPOT1 and P3N-PIPOT2 with 29 and 63 amino acids deleted from the C-terminus of PIPO, respectively, revealed that the 19 amino acids at the N-terminus of PIPO contributed to the PD localization. Interaction assays showed that the 63 amino acids at the C-terminus of PIPO determined the P3N-PIPO interaction with PM-associated Ca2+-binding protein 1, ScPCaP1, which was isolated from the SCMV-susceptible sugarcane cultivar Badila. Like wild-type P3N-PIPO, P3N-PIPOT1 and P3N-PIPOT2 could translocate to neighbouring cells and recruit the SCMV cylindrical inclusion protein to the PM. Thus, interactions with ScPCaP1 may contribute to, but not determine, SCMV Pm3N-PIPO's localization to the PM or PD. These results also imply the existence of truncated P3N-PIPO in nature.

Effects of Methyl Mercury Chloride on Rat Hippocampus Structure.

The objective of this study is to investigate the impacts of Methyl Mercury Chloride (MMC) on cognitive functions and ultrastructural changes of hippocampus in Sprague Dawley (SD) rats. Thirty healthy 20-day-old male SD rats weighing 30-40 g were randomly divided into three groups to receive daily injections. Two different dose levels were used: 4 mg/kg as high dose (H-MMC) and 2 mg/kg as low dose (L-MMC).The control group received 4 mg/kg saline solution (N-NaCl). After daily subcutaneous injection for 50 days, 6-day Morris water maze tests were used to assess the learning and memory functions of the rats. After a 5-day continuous training, spatial probe tests were conducted of times and paths crossing to the target quadrant on the 6th day. After the rats were euthanized, their hippocampus sections were stained with hematoxylin and eosin and analyzed under bothoptical microscope and electron microscope. The time H-MMC group spent in finding platform was significantly longer as compared toN-NaCl group on day 2 to day 5 and L-MMC group on day 4 to day 5. The number of crossing times of H-MMC group to the target quadrant was 0.63 ± 0.74, which is much lower than C-NaCl group (3.13 ± 1.56) with P value <0.05. No statistically significant difference in crossing times was found between L-MMC and C-NaCl groups. For H-MMC group, decreasing number of neurons and disorganized nerve cells were examined under light microscope. Swelling and dissolution of Golgi complex were examined under electron microscope, along with endoplasmic reticulum expansion and cytoplasmic edema. Mild cytoplasmic edema was found in L-MMC group. MMC can cause cognitive impairment in terms of learning and memory in SD rats. Additionally, it can also cause changes in the ultrastructure of neurons and morphological changes in the hippocampus, causing significant damage.

Sugarcane Elongin C is involved in infection by sugarcane mosaic disease pathogens.

Sugarcane (Saccharum sp. hybrid) provides the main source of sugar for humans. Sugarcane mosaic disease (SMD) is a major threat to sugarcane production. Currently, control of SMD is mainly dependent on breeding resistant cultivars through hybridization, which is time-consuming. Understanding the mechanism of viral infection may facilitate novel strategies to breed cultivars resistant to SMD and to control the disease. In this study, a wide interaction was detected between the viral VPg protein and host proteins. Several genes were screened from sugarcane cDNA library that could interact with Sugarcane streak mosaic virus VPg, including SceIF4E1 and ScELC. ScELC was predicted to be a cytoplasmic protein, but subcellular localization analysis showed it was distributed both in cytoplasmic and nuclear, and interactions were also detected between ScELC and VPg of SCMV or SrMV that reveal ScELC was widely used in the SMD pathogen infection process. ScELC and VPgs interacted in the nucleus, and may function to enhance the viral transcription rate. ScELC also interacted with SceIF4E2 both in the cytoplasm and nucleus, but not with SceIF4E1 and SceIF4E3. These results suggest that ScELC may be essential for the function of SceIF4E2, an isomer of eIF4E.

Methylmercury chloride damage to the adult rat hippocampus cannot be detected by proton magnetic resonance spectroscopy.

Previous studies have found that methylmercury can damage hippocampal neurons and accordingly cause cognitive dysfunction. However, a non-invasive, safe and accurate detection method for detecting hippocampal injury has yet to be developed. This study aimed to detect methylmercury-induced damage on hippocampal tissue using proton magnetic resonance spectroscopy. Rats were given a subcutaneous injection of 4 and 2 mg/kg methylmercury into the neck for 50 consecutive days. Water maze and pathology tests confirmed that cognitive function had been impaired and that the ultrastructure of hippocampal tissue was altered after injection. The results of proton magnetic resonance spectroscopy revealed that the nitrogen-acetyl aspartate/creatine, choline complex/creatine and myoinositol/creatine ratio in rat hippocampal tissue were unchanged. Therefore, proton magnetic resonance spectroscopy can not be used to determine structural damage in the adult rat hippocampus caused by methylmercury chloride.