Agrobacterium-mediated transformation of B. juncea reveals that BjuLKP2 functions in plant yellowing.

KEY MESSAGE: A stable Agrobacterium-mediated transformation system was constructed for B. juncea, and BjuLKP2 was overexpressed, leading to plant yellowing. A stable and efficient transformation system is necessary to verify gene functions in plants. To establish an Agrobacterium-mediated transformation system for B. juncea, various factors, including the explant types, hormone combination and concentration, infection time and concentration, were optimized. Eventually, a reliable system was established, and two BjuLKP2 overexpression (OE) lines, which displayed yellowing of cotyledons, shoot tips, leaves and flower buds, as well as a decrease in total chlorophyll content, were generated. qRT-PCR assays revealed significant upregulation of five chlorophyll synthesis genes and downregulation of one gene in the BjuLKP2 OE line. Furthermore, antioxidant capacity assays revealed reduced activities of APX, CAT and SOD, while POD activity increased in the BjuLKP2 OE26. Additionally, the kinetic determination of chlorophyll fluorescence induction suggested a decrease in the photosynthetic ability of BjuLKP2 OE26. GUS assays revealed the expression of BjuLKP2 in various tissues, including the roots, hypocotyls, cotyledons, leaf vasculature, trichomes, sepals, petals, filaments, styles and stigma bases, but not in seeds. Scanning electron revealed alterations in chloroplast ultrastructure in both the sponge and palisade tissue. Collectively, these findings indicate that BjuLKP2 plays a role in plant yellowing through a reduction in chlorophyll content and changes in chloroplasts structure.

Magnesium oxide nanoparticles reduce clubroot by regulating plant defense response and rhizosphere microbial community of tumorous stem mustard (Brassica juncea var. tumida).

Clubroot, caused by Plasmodiophora brassicae, is a major disease that significantly impairs the yield of cruciferous crops and causes significant economic losses across the globe. The prevention of clubroot, especially in tumorous stem mustard (without resistant varieties), are is limited and primarily relies on fungicides. Engineered nanoparticles have opened up new avenues for the management of plant diseases, but there is no report on their application in the prevention of clubroot. The results showed that the control efficacy of 500 mg/L MgO NPs against clubroot was 54.92%. However, when the concentration was increased to 1,500 and 2,500 mg/L, there was no significant change in the control effect. Compared with CK, the average fresh and dry weight of the aerial part of plants treated with MgO NPs increased by 392.83 and 240.81%, respectively. Compared with the F1000 treatment, increases were observed in the content of soil available phosphorus (+16.72%), potassium (+9.82%), exchangeable magnesium (+24.20%), and water-soluble magnesium (+20.64%) in the 1,500 mg/L MgO NPs treatment. The enzyme-linked immune sorbent assay (ELISA) results showed that the application of MgO NPs significantly increased soil peroxidase (POD, +52.69%), alkaline protease (AP, +41.21%), alkaline phosphatase (ALP, +79.26%), urease (+52.69%), and sucrase (+56.88%) activities; And also increased plant L-phenylalanine ammonla-lyase (PAL, +70.49%), polyphenol oxidase (PPO, +36.77%), POD (+38.30%), guaiacol peroxidase (POX, +55.46%) activities and salicylic acid (SA, +59.86%) content. However, soil and plant catalase (CAT, -27.22 and - 19.89%, respectively), and plant super oxidase dismutase (SOD, -36.33%) activities were significantly decreased after the application of MgO NPs. The metagenomic sequencing analysis showed that the MgO NPs treatments significantly improved the α-diversity of the rhizosphere soil microbial community. The relative abundance of beneficial bacteria genera in the rhizosphere soil, including Pseudomonas, Sphingopyxis, Acidovorax, Variovorax, and Bosea, was significantly increased. Soil metabolic functions, such as oxidative phosphorylation (ko00190), carbon fixation pathways in prokaryotes (ko00720), indole alkaloid biosynthesis (ko00901), and biosynthesis of various antibiotics (ko00998) were significantly enriched. These results suggested that MgO NPs might control clubroot by promoting the transformation and utilization of soil nutrients, stimulating plant defense responses, and enriching soil beneficial bacteria.

Plant Genotype Shapes the Soil Nematode Community in the Rhizosphere of Tomatoes with Different Resistance to Meloidognye incognita.

Soil nematodes are considered indicators of soil quality due to their immediate responses to changes in the soil environment and plants. However, little is known about the effects of plant genotypes on the soil nematode community. To elucidate this, high-throughput sequencing and gas chromatography/mass spectrometry analysis was conducted to analyze the soil nematode community and the structure of root exudates in the rhizosphere of tomatoes with different resistance to Meloidognye incognita. The dominant soil nematode group in the soil of resistant tomatoes was Acrobeloides, while the soil nematode group in the rhizosphere of the susceptible and tolerant tomatoes was Meloidognye. Hierarchical clustering analysis and non-metric multidimensional scaling showed that the three soil nematode communities were clustered into three groups according to the resistance level of the tomato cultivars. The soil nematode community of the resistant tomatoes had a higher maturity index and a low plant-parasite index, Wasilewska index and disease index compared to the values of the susceptible and tolerant tomatoes. Redundancy analysis revealed that the disease index and root exudates were strongly related to the soil nematode community of three tomato cultivars. Taken together, the resistance of the tomato cultivars and root exudates jointly shapes the soil nematode community. This study provided a valuable contribution to understanding the mechanism of plant genotypes shaping the soil nematode community.

[Response of Photosynthetic Bacterial Community to Cadmium Contamination in Paddy Soil].

Heavy metal pollution in rice fields leads to huge losses in rice yield every year and is thus of increasing concern. Therefore, it is important to understand the changes in the microecology and physicochemical properties of paddy soil under different levels of cadmium pollution. The purpose of this study was to investigate the response of the photosynthetic bacterial community in paddy soil to different cadmium pollution levels using 16S sequencing technology. The results showed that pH, total cadmium, and available cadmium content decreased gradually with the increase in cadmium pollution. The soil α diversity was slightly different in the high cadmium (Cd), medium Cd, and low Cd groups; however, the enriched photosynthetic populations and photosynthetic bacterial communities were significantly different among these groups. The effective connections between photosynthetic bacterial species in the high Cd group were significantly greater than those in the medium and low Cd groups, the connections were closer, and the density was higher. Alkaline nitrogen, pH, available (P/K), total (N/P), organic matter, total cadmium, and available cadmium were important factors affecting the photosynthetic bacterial community and were significantly correlated with the photosynthetic bacterial community, explaining 59.90% of the variation in the photosynthetic bacterial community. Effective Cd content was significantly positively correlated with Methylorubrum populi, Methylorubrum extorquens, Methylobacterium sp. Leaf125, and Rhodopseudomonas sp. AAP120 (R>0.05, P<0.05). This study will provide a theoretical basis for the microbial remediation of cadmium contamination in paddy fields. This study is important for understanding the effects of cadmium pollution on specific functional microbial populations in paddy soils.

Exploration of Intrinsic Microbial Community Modulators in the Rice Endosphere Indicates a Key Role of Distinct Bacterial Taxa Across Different Cultivars.

Microbial communities associated with the plant phyllosphere and endosphere can have both beneficial as well as detrimental effects on their hosts. There is an ongoing debate to which extend the phyllosphere and endosphere microbiome assembly is controlled by the host plant how pronounced cultivar effects are. We investigated the bacterial and fungal communities from the phyllosphere and endosphere of 10 different rice cultivars grown under identical environmental conditions in the frame of a targeted approach to identify drivers of community assembly. The results indicated that the endophytic bacterial communities were clearly separated into two groups. The α-diversity and microbial network complexity within Group I were significantly lower than in Group II. Moreover, the genera Nocardioides, Microvirga, and Gaiella were significantly more abundant in Group II and only present in the interaction networks of this group. These three genera were significantly correlated with α- and ß-diversity of the endophytic bacterial community and thus identified as major drivers of the endosphere community. We have identified keystone taxa that shape endophytic bacterial communities of different rice cultivars. Our overall findings provide new insights into plant-microbe interactions, and may contribute to targeted improvements of rice varieties in the future.

Genome-wide identification and analysis of NPR family genes in Brassica juncea var. tumida.

Nonexpressor of pathogenesis-related (NPR) genes are bona fide transcription cofactors in the signal transduction pathway of salicylic acid (SA) and play critical regulatory roles in plant immunity. However, the NPR family genes in Brassica juncea var. tumida have not yet been comprehensively identified and analyzed as of yet. In the present study, NPR genes in B. juncea var. tumida seedlings were identified, and the tissue-specific expression patterns of NPR genes in the seedling were analyzed under salt stress (200 mM) treatment and infection by Plasmodiophora brassicae. A total of 19 NPR family genes clustering into six separate groups were identified in the genome of B. juncea var. tumida. These BjuNPR family genes were located in 11 of 18 chromosomes of B. juncea var. tumida and each possessed 1-5 exons. The BjuNPR family members had similar protein structures and conserved motifs. The BjuNPR genes exhibited tissue-specific expression patterns in the root, stem, leaf, flower and pod. Some BjuNPR genes were sensitive to salt stress and showed up-regulated or down-regulated expression patterns and most BjuNPR genes were up-regulated upon infection by P. brassicae. This study provides a foundation for further research into BjuNPR genes regulation in plant growth, development, and abiotic stress tolerance.

Physiological change alters endophytic bacterial community in clubroot of tumorous stem mustard infected by Plasmodiophora brassicae.

BACKGROUND: Endophytic bacteria are considered as symbionts living within plants and are influenced by abiotic and biotic environments. Pathogen cause biotic stress, which may change physiology of plants and may affect the endophytic bacterial communiy. Here, we reveal how endophytic bacteria in tumorous stem mustard (Brassica juncea var. tumida) are affected by plant physiological changes caused by Plasmodiophora brassicae using 16S rRNA high-throughput sequencing. RESULTS: The results showed that Proteobacteria was the dominant group in both healthy roots and clubroots, but their abundance differed. At the genus level, Pseudomonas was dominant in clubroots, whereas Rhodanobacter was the dominant in healthy roots. Hierarchical clustering, UniFrac-weighted principal component analysis (PCA), non-metric multidimensional scaling (NMDS) and analysis of similarities (ANOSIM) indicated significant differences between the endophytic bacterial communities in healthy roots and clubroots. The physiological properties including soluble sugar, soluble protein, methanol, peroxidase (POD) and superoxide dismutase (SOD) significantly differed between healthy roots and clubroots. The distance-based redundancy analysis (db-RDA) and two-factor correlation network showed that soluble sugar, soluble protein and methanol were strongly related to the endophytic bacterial community in clubroots, whereas POD and SOD correlated with the endophytic bacterial community in healthy roots. CONCLUSIONS: Our results illustrate that physiologcial changes caused by P. brassicae infection may alter the endophytic bacterial community in clubroots of tumorous stem mustard.

Genome-wide identification and expression analysis of jasmonate ZIM domain gene family in tuber mustard (Brassica juncea var. tumida).

Tuber mustard, which is the raw material of Fuling pickle, is a crop with great economic value. However, during growth and development, tuber mustard is frequently attacked by the pathogen Plasmodiophora brassicae and frequently experiences salinity stress. Jasmonic acid (JA) is a hormone related to plant resistance to biotic and abiotic stress. Jasmonate ZIM domain proteins (JAZs) are crucial components of the JA signaling pathway and play important roles in plant responses to biotic and abiotic stress. To date, no information is available about the characteristics of the JAZ family genes in tuber mustard. Here, 38 BjJAZ genes were identified in the whole genome of tuber mustard. The BjJAZ genes are located on 17 of 18 chromosomes in the tuber mustard genome. The gene structures and protein motifs of the BjJAZ genes are conserved between tuber mustard and Arabidopsis. The results of qRT-PCR analysis showed that BjuA030800 was specifically expressed in root, and BjuA007483 was specifically expressed in leaf. In addition, 13 BjJAZ genes were transiently induced by P. brassicae at 12 h, and 7 BjJAZ genes were induced by salt stress from 12 to 24 h. These results provide valuable information for further studies on the role of BjJAZ genes in the regulation of plant growth and development and in the response to biotic and abiotic stress.

Genome-wide identification and gene expression analysis of SOS family genes in tuber mustard (Brassica juncea var. tumida).

The Salt Overly Sensitive (SOS) pathway in Arabidopsis thaliana plays important roles in maintaining appropriate ion homeostasis in the cytoplasm and regulating plant tolerance to salinity. However, little is known about the details regarding SOS family genes in the tuber mustard crop (Brassica juncea var. tumida). Here, 12 BjSOS family genes were identified in the B. juncea var. tumida genome including two homologous genes of SOS1, one and three homologs of SOS2 and SOS3, two homologs of SOS4, two homologs of SOS5 and two homologs of SOS6, respectively. The results of conserved motif analysis showed that these SOS homologs contained similar protein structures. By analyzing the cis-elements in the promoters of those BjSOS genes, several hormone- and stress-related cis-elements were found. The results of gene expression analysis showed that the homologous genes were induced by abiotic stress and pathogen. These findings indicate that BjSOS genes play crucial roles in the plant response to biotic and abiotic stresses. This study provides valuable information for further investigations of BjSOS genes in tuber mustard.

Infection of Plasmodiophora brassicae changes the fungal endophyte community of tumourous stem mustard roots as revealed by high-throughput sequencing and culture-dependent methods.

Diverse fungal endophytes live in plants and are shaped by some abiotic and biotic stresses. Plant disease as particular biotic stress possibly gives an impact on the communities of fungal endophytes. In this study, clubroot disease caused by an obligate biotroph protist, Plasmodiophora brassicae, was considered to analyze its influence on the fungal endophyte community using an internal transcribed spacer (ITS) through high-throughput sequencing and culture-dependent methods. The results showed that the diversity of the endophyte community in the healthy roots was much higher than the clubroots. Ascomycota was the dominant group of endophytes (Phoma, Mortierella, Penicillium, etc.) in the healthy roots while P. brassicae was the dominant taxon in the clubroots. Hierarchical clustering, principal component analysis (PCA), principal coordinates analysis (PCoA) and analysis of similarities (ANOSIM) indicated significant differences between the endophyte communities in the healthy roots and clubroots. Linear discriminant analysis effect size (LefSe) analysis showed that the dominant genera could be regarded as potential biomarkers. The endophyte community in the healthy roots had a more complex network compared with the clubroots. Also, many plant pathogenic Fusarium were isolated from the clubroots by the culture-dependent method. The outcome of this study illustrates that P. brassicae infection may change the fungal endophyte community associated with the roots of tumourous stem mustard and facilitates the entry of soil pathogen into the roots.

Genome-Wide Analysis of Auxin Receptor Family Genes in Brassica juncea var. tumida.

Transport inhibitor response 1/auxin signaling f-box proteins (TIR1/AFBs) play important roles in the process of plant growth and development as auxin receptors. To date, no information has been available about the characteristics of the TIR1/AFB gene family in Brassica juncea var. tumida. In this study, 18 TIR1/AFB genes were identified and could be clustered into six groups. The genes are located in 11 of 18 chromosomes in the genome of B. juncea var. tumida, and similar gene structures are found for each of those genes. Several cis-elements related to plant response to phytohormones, biotic stresses, and abiotic stresses are found in the promoter of BjuTIR1/AFB genes. The results of qPCR analysis show that most genes have differential patterns of expression among six tissues, with the expression levels of some of the genes repressed by salt stress treatment. Some of the genes are also responsive to pathogen Plasmodiophora brassicae treatment. This study provides valuable information for further studies as to the role of BjuTIR1/AFB genes in the regulation of plant growth, development, and response to abiotic stress.

The Soybean GmNARK Affects ABA and Salt Responses in Transgenic Arabidopsis thaliana.

GmNARK (Glycine max nodule autoregulation receptor kinase) is the homolog of Arabidopsis thaliana CLAVATA1 (CLV1) and one of the most important regulators in the process of AON (Autoregulation of Nodulation), a process that restricts excessive nodule numbers in soybean. However, except for the function in AON, little is known about this gene. Here, we report that GmNARK plays important roles in process of plant response to abiotic stresses. Bioinformatic analysis and subcellular localization experiment results showed that GmNARK was a putative receptor like kinase and located at membrane. The promoter of GmNARK contains manifold cis regulatory elements that are responsive to hormone and stresses. Gene transcript expression pattern analysis in soybean revealed GmNARK was induced by ABA and NaCl treatment in both shoot and root. Overexpression of GmNARK in Arabidopsis resulted in higher sensitivity to ABA and salt treatment during seed germination and greening stages. We also checked the expression levels of some ABA response genes in the transgenic lines; the results showed that the transcript level of all the ABA response genes were much higher than that of wild type under ABA treatment. Our results revealed a novel role of GmNARK in response to abiotic stresses during plant growth and development.

Double-stranded RNA-mediated interference of dumpy genes in Bursaphelenchus xylophilus by feeding on filamentous fungal transformants.

RNA interference (RNAi) is a valuable tool for studying gene function in vivo and provides a functional genomics platform in a wide variety of organisms. The pinewood nematode, Bursaphelenchus xylophilus, is a prominent invasive plant-parasitic nematode and has become a serious worldwide threat to forest ecosystems. Presently, the complete genome sequence of B. xylophilus has been published, and research involving genome-wide functional analyses is likely to increase. In this study, we describe the construction of an effective silencing vector, pDH-RH, which contains a transcriptional unit for a hairpin loop structure. Utilising this vector, double-stranded (ds)RNAs with sequences homologous to the target genes can be expressed in a transformed filamentous fungus via Agrobacterium tumefaciens-mediated transformation technology, and can subsequently induce the knockdown of target gene mRNA expression in B. xylophilus by allowing the nematode to feed on the fungal transformants. Four dumpy genes (Bx-dpy-2, 4, 10 and 11) were used as targets to detect RNAi efficiency. By allowing the nematode to feed on target gene-transformed Fusarium oxysporum strains, target transcripts were knocked down 34-87% compared with those feeding on the wild-type strain as determined by real-time quantitative PCR (RT-qPCR). Morphological RNAi phenotypes were observed, displaying obviously reduced body length; weak dumpy or small (short and thin) body size; or general abnormalities. Moreover, compensatory regulation and non-specific silencing of dpy genes were found in B. xylophilus. Our results indicate that RNAi delivery by feeding in B. xylophilus is a successful technique. This platform may provide a new opportunity for undertaking RNAi-based, genome-wide gene functional studies in vitro in B. xylophilus. Moreover, as B. xylophilus feeds on endophytic fungi when a host has died, RNAi feeding technology will offer the prospect for developing a novel control strategy for the nematode. Furthermore, this platform may also be applicable to other parasitic nematodes that have a facultative, fungivorous habit.

Preliminary research on the expression, purification and function of the apoptotic fusion protein, Sival.

The objective of the present study was to investigate cloning, expression, and functions of the recombinant protein, Siva1. Siva1 gene was synthesized by RT-PCR from HCT116 cells. Plasmids were cleaved with the restriction endonuclease, BamH1/Sal1 and products were connected to pQE30, which underwent cleavage by BamH1/Sal1. The recombinant plasmid, pQE30-Siva1, was identified after digestion with restriction endonucleases followed by transformation into E. coli M15. Expression of Siva1 was induced by IPTG and identified by SDS- PAGE following purification with affinity chromatography. The results showed that size of Siva1 was 12 kDa, consistent with the molecular weight of the His-Siva1 fusion protein. Functional test demonstrated that Siva1 significantly inhibited the invasion and migration of HCT116 cells. It may thus find clinical application for control of cancers.

[Experimental study of HIF-1alpha antisense oligonucleotide on implanted human gastric cancer in nude mice].

OBJECTIVE: To observe the effect of HIF-1alpha antisense oligonucleotide (HIF-1alpha ASOND) on implanted human gastric cancer in nude mice. METHODS: BALB/c nude mice were subcutaneously transplanted with SCG-7901 tumor cells, then the mice were randomly divided into antisense oligonucleotide (ASOND) group, sense oligonucleotide (SOND) group and the control group. The HIF-1alpha ASOND complexed with cationic liposome, HIF-1alpha SOND complexed with cationic liposome and liposome were injected intra-tumorally in the above groups, respectively. Tumor growth curve in each group animals was observed. The tumor weight was measured and then the rate of inhibition was calculated. The morphological changes of tumor cells was observed under microscope. The expression of HIF-1alpha, VEGF and MVD in tumor tissue was determined by immunohistochemical methods. RESULTS: The growth of tumor of ASOND group was significantly inhibited. There was statistically significant difference (P < 0.05) in the weight of tumor between ASOND group and control group. The inhibitory rates of ASOND group and SOND group were 47.94% and 16.88% respectively, and both inhibitory rates were significantly different (P < 0.01). The tumor tissue expression of HIF-1alpha, VEGF and MVD in ASOND group was significantly lower than those of SOND and the control groups (P < 0.01). CONCLUSION: In tumor tissue, the injection of HIF-1alpha ASOND to treat gastric carcinoma transplanted subcutaneously in nude mice can reduce the expression of HIF-1alpha, consequently reduce the formation of VEGF and new blood vessels, so as to achieve inhibition of tumor growth.