SLC10A3 Is a Prognostic Biomarker and Involved in Immune Infiltration and Programmed Cell Death in Lower Grade Glioma.

BACKGROUND: The association between SLC10A3 (solute carrier family 10 member 3) and lower grade glioma (LGG) remains unclear. METHODS: We used public databases and bioinformatics analysis to analyze SLC10A3. These included The Cancer Genome Atlas, Genotype-Tissue Expansion, Chinese Glioma Genome Atlas, Human Protein Atlas, GeneCards, cBioPortal, Search Tool for the Retrieval of Interacting Genes/Proteins, Gene Expression Profiling Interactive Analysis, Tumor Immune Estimation Resource, Tumor-Immune System Interaction Database, receiver operating characteristic curve analysis, Kaplan-Meier analysis, Cox analysis, nomograms, calibration plots, gene ontology/Kyoto Encyclopedia of Genes and Genomes enrichment analysis, gene set enrichment analysis, single-sample gene set enrichment analysis, and Spearman's correlation analysis. RESULTS: SLC10A3 was upregulated in adrenocortical carcinoma, glioblastoma, and LGG and was associated with good overall survival (OS) in adrenocortical carcinoma and poor OS in LGG and glioblastoma. SLC10A3 was increased with increased World Health Organization grade, upregulated in isocitrate dehydrogenase-wild type, 1p/19q (chromosome arms 1p and 19q) non-co-deleted, and higher in astrocytoma. Patients with LGG were grouped by the occurrence of the clinical outcome endpoints (i.e., OS, disease-specific survival [DSS], and progression-free interval events). Genetic alterations in SLC10A3 were associated with poor progression-free survival in LGG. Most of clinical characteristics were associated with the SLC10A3 expression level. SLC10A3 with diagnostic and prognostic value (OS, DSS, and progression-free interval) was an independent prognostic factor in LGG. Moreover, Nomograms (WHO grade, 1p/19q codeletion, age and SLC10A3) had moderately accurate predictive for OS and DSS. Functional analysis showed that SLC10A3 might participate in the transport of multiple substances, neurogenic signaling, immune response, and programmed cell death in LGG. SLC10A3 correlated with immune infiltration in LGG and moderately correlated with the gene signature of pyroptosis, lysosome-dependent cell death, necroptosis, apoptosis, ferroptosis, alkaliptosis, and autophagy-dependent cell death. CONCLUSIONS: SLC10A3 is a potential diagnostic and prognostic biomarker for LGG and might be associated with substance transport, neurogenic signaling, immune infiltration, and programmed cell death in LGG.

Multi-Omics Analysis Reveals the Resistance Mechanism and the Pathogens Causing Root Rot of Coptis chinensis.

Coptis chinensis is a traditional Chinese medicinal herb used for more than 2,000 years. Root rot in C. chinensis can cause brown discoloration (necrosis) in the fibrous roots and rhizomes, leading to plants wilting and dying. However, little information exists about the resistance mechanism and the potential pathogens of the root rot of C. chinensis plants. As a result, in order to investigate the relationship between the underlying molecular processes and the pathogenesis of root rot, transcriptome and microbiome analyses were performed on healthy and diseased C. chinensis rhizomes. This study found that root rot can lead to the significant reduction of medicinal components of Coptis, including thaliotrine, columbamine, epiberberin, coptisine, palmatine chloride, and berberine, affecting its efficacy quality. In the present study, Diaporthe eres, Fusarium avenaceum, and Fusarium solani were identified as the main pathogens causing root rot in C. chinensis. At the same time, the genes in phenylpropanoid biosynthesis, plant hormone signal transduction, plant-pathogen interaction, and alkaloid synthesis pathways were involved in the regulation of root rot resistance and medicinal component synthesis. In addition, harmful pathogens (D. eres, F. avenaceum and F. solani) also induce the expression of related genes in C. chinensis root tissues to reduce active medicinal ingredients. These results provide insights into the root rot tolerance study and pave the way for process disease resistance breeding and quality production of C. chinensis. IMPORTANCE Root rot disease significantly reduces the medicinal quality of Coptis chinensis. In the present study, results found that the C. chinensis fibrous and taproot have different tactics in response to rot pathogen infection. Diaporthe eres, Fusarium avenaceum, and Fusarium solani were isolated and identified to cause different degrees of C. chinensis root rot. These results are helpful for researchers to further explore the mechanism of resistance to rhizoma Coptis root rot.

OsWRKY93 Dually Functions Between Leaf Senescence and in Response to Biotic Stress in Rice.

Cross talking between natural senescence and cell death in response to pathogen attack is an interesting topic; however, its action mechanism is kept open. In this study, 33 OsWRKY genes were obtained by screening with leaf aging procedure through RNA-seq dataset, and 11 of them were confirmed a significant altered expression level in the flag leaves during aging by using the reverse transcript quantitative PCR (RT-qPCR). Among them, the OsWRKY2, OsWRKY14, OsWRKY26, OsWRKY69, and OsWRKY93 members exhibited short-term alteration in transcriptional levels in response to Magnaporthe grisea infection. The CRISPR/Cas9-edited mutants of five genes were developed and confirmed, and a significant sensitivity to M. oryzae infection was observed in CRISPR OsWRKY93-edited lines; on the other hand, a significant resistance to M. oryzae infection was shown in the enhanced expression OsWRKY93 plants compared to mock plants; however, enhanced expression of other four genes have no significant affection. Interestingly, ROS accumulation was also increased in OsWRKY93 enhanced plants after flg22 treatment, compared with the controls, suggesting that OsWRKY93 is involved in PAMP-triggered immune response in rice. It indicated that OsWRKY93 was involved in both flag leaf senescence and in response to fungi attack.

First report of Diaporthe eres causing root rot of Coptis chinensis Franchet.

Chongqing coptis (Coptis chinensis Franchet) industry produces more than 60% of the Chinese coptis crop, and has been exported to many countries and regions. Since 2008, root rot has become a serious and widespread disease on coptis plants in Shizhu county with an average incidence of 40%, and yield losses up to 67%. Symptomatic coptis plants showed stunted growth, with the fibrous roots and main roots having brown or black, rotten, necrotic lesions. To our knowledge, Fusarium solani, F. carminascens, F. oxysporum and F. tricinctum have been previously reported as pathogens of coptis root rot (Luo et al. 2014; Cheng et al. 2020; Wu et al. 2020), but non Fusarium pathogens has not been reported yet. In order to identify new pathogens, 33 diseased roots were collected from Shizhu (30°18'N, 108°30'E) in October 2019. Small samples (0.5 cm in length) were cut from the border between diseased and healthy tissue, and then put on PDA after surface sterilization. Cultures were incubated at 25°C in dark until fungal colonies were observed. After subculturing for 3 times, 3 out of 21 isolates yielded a similar type of fungal colony. White, aerial, fluffy mycelium were formed and reached 8.3 cm diameter within 7 days, and dark pigmentation developed in the centre. Colonies turned to gray with age, and abundant dark brown pycnidia and black stromata were formed at maturity. Alpha conidia were aseptate, hyaline, fusiform to ellipsoidal, often biguttulate, measuring (6.0-8.5)×(2.0-3.0) µm. Beta conidia were aseptate, hyaline, linear to hooked, measuring (18-30)×(1.0-1.5) µm (Figure S1). For further identification, a multigene phylogenetic analysis was carried out. The internal transcribed spacer (ITS), translation elongation factor 1ɑ (tef1-ɑ), histone H3 (his3), calmodulin (cal), and ß-tubulin (tub2) gene regions were amplified with ITS1/ITS4, EF1-728F/EF1-986R, CYLH3F/H3-1b, CAL228F/CAL737R, T1/Bt2b (White et al. 1990; Glass and Donaldson 1995; Carbone and Kohn 1999; Crous et al. 2004). GenBank accession numbers of isolate H13 were MT463391 for the ITS region, MT975573 for tef1-ɑ, MT975574 for his3, MT975575 for cal, and MT975576 for tub2. BLAST results showed the ITS, tef1-ɑ, his3, cal and tub2 sequences revealed 99.82% (553/554 base pairs), 100% (347/347 base pairs), 100% (474/474 base pairs), 99.39% (486/489 base pairs), and 99.14% (803/810 base pairs) homology respectively with those of Diaporthe eres (MN816416.1, KU557616.1, KC343564.1, KU557595.1, and KY569366.1). Thus, H13 were identified as D. eres based on its morphological and molecular characteristics. Pathogenicity of D. eres in coptis was investigated using the H13 isolate (1 of the 3 isolates). The roots of 10 healthy 2-year-old coptis plants were individually inoculated with 5 ml of a 106 conidia/mL conidial suspension and sterilized water was used to mock inoculate. Thirty days after inoculation, most of the inoculated coptis roots showed dark brown and rotten root, similar to those observed in the field, whereas mock inoculated roots showed healthy. D. eres was recovered from symptomatic roots and identified based on morphology. To our knowledge, this is the first report of D. eres causing root rot of coptis not only in China but anywhere in the world.

First Report of Root Rot Caused by Fusarium avenaceum on Coptis chinensis Franchet in Chongqing, China.

Coptis chinensis Franchet, is a perennial herb used as a traditional Chinese medicine. Annual production of Coptis is about 3000 tons in Shizhu, Chongqing. In recent years, root rot has become a serious and widespread disease on Coptis in Shizhu with an average incidence of 40%, and yield losses up to 67%. Infected plants were easy to pull from the soil, and most of the fibrous roots and main roots were brown or black compared to healthy roots that were yellow. Severely infected plants were wilted and necrotic. In October 2019, 33 diseased roots were collected from Shizhu (30°18'N, 108°30'E), and small samples (0.5 cm in length) were cut from the border between diseased and healthy tissue, successively sterilized with 75% ethanol and 2% sodium hypochlorite, rinsed 3 times with sterilized water, dried on sterilized filter paper, and transferred onto PDA, and incubated at 25°C for 7 days in dark. Eighteen distinct fungal isolates (H1-H18) were isolated and Koch's postulates were conducted to verify the pathogenicity of individual isolates. The rhizosphere soil of healthy 2-year-old Coptis plants was inoculated by pouring 5 mL of conidial suspension (106 conidia/mL) scraped from a culture of each isolate on PDA. Sterilized water was used to mock inoculate. For each isolate, 6 plants were inoculated. After 20 days, the roots of all plants inoculated with H15 or H18 were dark brown and rotten, while mock inoculated plants were healthy. The isolates H15 and H18 were re-isolated from symptomatic plants. Isolate H15 formed abundant white mycelium on PDA and produced rose pigment in the agar. Conidia were long and slender, straight to slightly curved, with 1-3 septate. The apical cells were tapering and bent, and the foot cells were distinctly notched. Conidiogenous cells were monophialides and polyphialides. No chlamydospores were observed (Figure S1). Isolate H18 formed white sparse mycelium on PDA and produced no pigment in the agar. Conidia were relatively wide, straight and stout, with 3-5 septate. The apical cells were blunt and rounded, and the foot cells were barely notched. Conidiogenous cells were long monophialides. Chlamydospores were formed intercalary in the hyphae (Figure S2). For further identification, the internal transcribed spacer (ITS), ß-tubulin, translation elongation factor 1ɑ (EF1ɑ) and RNA polymerase second largest subunit (RPB2) gene regions were amplified with ITS1/ITS4, Bt2a/Bt2b, EF1/EF2 and 5f2/7cr (White et al. 1990; Glass and Donaldson, 1995; O'Donnell et al. 2010). GenBank accession numbers of H15 and H18 were MT463390 and MT463389 for the ITS region, MT465656 and MT465654 for ß-tubulin, MT653321 and MT465651 for EF1ɑ, and MT653323 and MT653322 for RPB2. BLAST results showed the ITS, ß-tubulin, EF1ɑ, and RPB2 sequences revealed 100% (533/533 base pairs), 100% (265/265 base pairs), 98% (622/632 base pairs), and 99% (936/947 base pairs) homology respectively with those of Fusarium avenaceum (MN186746.1, MH791368.1, KU238140.1, and MK185027.1), and 100% (537/537 base pairs), 100% (227/227 base pairs), 100% (688/688 base pairs), and 99.03% (918/927 base pairs) with F. solani in GenBank (MH857319.1, MN692929.1, KP674211.1, and MH300549.1), respectively. Thus, H15 and H18 were identified as F. avenaceum and F. solani based on its morphological and molecular characteristics. To our knowledge, F. solani has been previously reported as a pathogen on Coptis (Luo et al. 2014), and this is the first report of root rot on Coptis caused by F. avenaceum in the world. Identification of the pathogens is important for effective disease management and control.

Functional study of Csrbohs in defence response against Xanthomonas citri ssp. citri.

NADPH oxidases, encoded by rbohs (respiratory burst oxidase homologues), transfer electrons from NADPH to molecular oxygen (O2) to generate superoxide anion (O2•-), which is the first step in the formation of hydrogen peroxide (H2O2) in the plant-pathogen interaction system. In the present work, six citrus rbohs (Csrbohs) genes were identified in citrus, and their possible involvement in resistance to Xanthomonas citri ssp. citri (Xcc) was examined. Inoculation with Xcc promoted the H2O2 production and induced expression of the Csrbohs, especially CsrbohD. Results showed that CsrbohD was markedly induced in the resistant genotype kumquat 'Luofu' [Fortunella margarita (Lour.) Swingle] compared with grapefruit 'Duncan' [Citrus paradisi (Linn.) Macf.]. Virus-induced gene silencing (VIGS) of CsrbohD resulted in reduced resistance to Xcc in grapefruit, but not in kumquat. Compared with non-silenced plants, canker-like symptoms were observed earlier, and they were more extensive in the CsrbohD-silenced grapefruit. Silencing of CsrbohD also suppressed the Xcc induced reactive oxygen species (ROS) burst, and resulted in accumulation of more Xcc bacterial colonies. Taken together, these data indicate that CsrbohD promotes resistance to Xcc, especially in grapefruit.

Development of Infectious cDNA Clones of Citrus Yellow Vein Clearing Virus Using a Novel and Rapid Strategy.

Yellow vein clearing disease (YVCD) causes significant economic losses in lemon and other species of citrus. Usually, citrus yellow vein clearing virus (CYVCV) is considered to be the causal agent of YVCD. However, mixed infection of CYVCV and Indian citrus ringspot virus (ICRSV) or other pathogens is often detected in citrus plants with YVCD. In this study, we re-examined the causal agent of YVCD to fulfill Koch's postulates. First, the full-length genome of CYVCV isolate AY (CYVCV-AY) was amplified by long-distance RT-PCR from a Eureka lemon (Citrus limon) tree with typical YVCD symptoms. The genomic cDNAs were then cloned into a ternary Yeast-Escherichia coli-Agrobacterium tumefaciens shuttle vector, pCY, using transformation-associated recombination (TAR) strategy, and 15 full-length cDNA clones of CYVCV-AY were obtained. Subsequently, four of these clones were selected randomly and inoculated on Jincheng (C. sinensis) seedlings through Agrobacterium-mediated vacuum-infiltration, and it was found that 80 to 100% of inoculated plants were infected with CYVCV by RT-PCR at 20 to 40 days postinoculation (dpi) and by direct tissue blot immunoassay at 60 dpi. The progeny of CYVCV-AY from cDNA clones caused typical symptoms of YVCD such as yellow vein clearing, leaf distortion, and chlorosis, which were the same as that elicited by wild-type virus. Finally, the regeneration of CYVCV-AY genome was confirmed by long-distance RT-PCR in lemon trees inoculated with the infectious cDNA clone. These results proved that CYVCV was the primary causal agent of YVCD. This is the first report on the development of infectious cDNA clones of CYVCV, which lays the foundation for further studies on viral gene functions and virus-host interactions.