Integrated Analyses of miRNome and Transcriptome Reveal the Critical Role of miRNAs Toward Heat Stress Response in Isochrysis galbana.

Isochrysis galbana is widely used in aquaculture as a bait microalgal species. High temperature (HT) can severely impair the development of I. galbana, exerting adverse effects on its yield. MicroRNAs (miRNAs) play an essential role in modulating stress-responsive genes. However, the role of miRNAs in response to HT in microalgae remains largely unexplored. In the present study, we identified several conserved and novel miRNAs in I. galbana through miRNome sequencing. Among these identified miRNAs, 22 miRNAs were differentially expressed in response to heat stress, and their target genes were predicted accordingly. Moreover, a comprehensive and integrated analysis of miRNome and transcriptome was performed. We found that six potential reversely correlated differentially expressed miRNA (DEM) and differentially expressed gene (DEG) pairs were associated with heat stress response (HSR) in I. galbana. The expressions of DEMs and DEGs were further verified using real-time quantitative PCR (RT-qPCR). Integrated analyses showed that miRNAs played fundamental roles in the regulatory network of HSR in I. galbana mainly by regulating some heat-responsive genes, including heat shock proteins (HSPs), reactive oxygen species (ROS) signaling-related genes, and specific key genes in the ubiquitination pathway. Our current study identified the first set of heat-responsive miRNAs from I. galbana and helped elucidate the miRNA-mediated HSR and resistance mechanisms in I. galbana. This new knowledge could provide ways to enhance its heat stress tolerance.

Responses of coastal sediment phosphorus release to elevated urea loading.

Increased urea is one of the common nitrogen forms polluting coastal waters and affecting nutrient dynamics. To investigate the effects of urea on sediment phosphorus (P) release, we carried out a 2-month mesocosm experiment with six targeted loadings of urea (0-0.6 mg N L-1 d-1). Results showed that: i) urea was rapidly transformed into ammonium and then nitrate (NO3-). ii) When nitrogen occurred as urea or ammonium, minor P release was observed. iii) After urea were mostly converted to NO3-, P release became clearer. iv) NO3- had a dual effect by promoting P release through decreasing sediment pH and increasing alkaline phosphatase activity or by inhibiting P release through improving sediment oxidation. v) The overall effects of urea on P release depended on the ultimate NO3- concentrations, being prominent when NO3- ≥ 11 mg N L-1. Our findings are of relevance when determining nitrogen reduction targets needed for combating eutrophication.

RNA-seq Insights Into the Impact of Alteromonas macleodii on Isochrysis galbana.

Phycospheric bacteria may be the key biological factors affecting the growth of algae. However, the studies about interaction between Isochrysis galbana and its phycospheric bacteria are limited. Here, we show that a marine heterotrophic bacterium, Alteromonas macleodii, enhanced the growth of I. galbana, and inhibited non-photochemical quenching (NPQ) and superoxide dismutase (SOD) activities of this microalgae. Further, we explored this phenomenon via examining how the entire transcriptomes of I. galbana changed when it was co-cultured with A. macleodii. Notable increase was observed in transcripts related to photosynthesis, carbon fixation, oxidative phosphorylation, ribosomal proteins, biosynthetic enzymes, and transport processes of I. galbana in the presence of A. macleodii, suggesting the introduction of the bacterium might have introduced increased production and transport of carbon compounds and other types of biomolecules. Besides, the transcriptome changed largely corresponded to reduced stress conditions for I. galbana, as inferred from the depletion of transcripts encoding DNA repair enzymes, superoxide dismutase (SOD) and other stress-response proteins. Taken together, the presence of A. macleodii mainly enhanced photosynthesis and biosynthesis of I. galbana and protected it from stress, especially oxidative stress. Transfer of fixed organic carbon, but perhaps other types of biomolecules, between the autotroph and the heterotroph might happen in I. galbana-A. macleodii co-culture. The present work provides novel insights into the transcriptional consequences of I. galbana of mutualism with its heterotrophic bacterial partner, and mutually beneficial associations existing in I. galbana-A. macleodii might be explored to improve productivity and sustainability of aquaculture algal rearing systems.

Integrated miRNAome and Transcriptome Analysis Reveals Argonaute 2-Mediated Defense Responses Against the Devastating Phytopathogen Sclerotinia sclerotiorum.

Argonaute 2 (AGO2)-mediated role in plant defense against fungal pathogens remains largely unknown. In this study, integrated miRNAome and transcriptome analysis employing ago2 mutant was performed to reveal AGO2-associated miRNAs and defense responses against the devastating necrotrophic phytopathogen Sclerotinia sclerotiorum. Both miRNAome and transcriptomes of S. sclerotiorum-inoculated ago2-1 mutant (ago2-Ss) and wild-type (WT-Ss) as well as mock-inoculated ago2-1 mutant (ago2) and wild-type (WT) Arabidopsis plants, were analyzed by sRNA and mRNA deep sequencing. Differentially expressed genes (DEGs) and differentially expressed miRNAs (DEMs) of the comparisons WT-Ss/WT, ago2/WT, ago2-Ss/WT-Ss, and ago2-Ss/ago2 were identified. Furthermore, integration analysis for the DEMs and DEGs identified over 40 potential AGO2-dependent Sclerotinia sclerotiorum-responsive (ATSR) DEM-DEG pairs involving modulation of immune recognition, calcium flux, redox homeostasis, hormone accumulation and signaling, cell wall modification and metal ion homeostasis. Data-mining result indicated that most of the DEMs were bound with AGO2. Moreover, Arabidopsis mutant analysis demonstrated that three ROS and redox homeostatasis related DEGs of identified DEM-DEG pairs, GSTU2, GSTU5, and RBOHF contributed to the AGO2-mediated defense against S. sclerotiorum. This work provides genome-wide prediction of miRNA-target gene pairs that are potentially associated with the AGO2-dependent resistance against S. sclerotiorum.

Bacterial Community Diversity and Screening of Growth-Affecting Bacteria From Isochrysis galbana Following Antibiotic Treatment.

Algal cultures are generally co-cultures of algae and bacteria, especially when considering outdoor cultivation. However, the effects of associated bacteria on algal growth remain largely unexplored, particularly in the context of Isochrysis galbana. In the present study, we investigated the effects of antibiotic on the growth of I. galbana and its associated bacterial community. We found advantageous responses of I. galbana to antibiotic exposure, evidenced by the increased growth, and the maximal photochemical efficiency of PSII (Fv/Fm). Since antibiotics can cause major disturbances within bacterial community, we further conducted 16S rDNA amplicon sequencing to determine the changes of bacterial community diversity following antibiotic treatment. We found that antibiotic treatment considerably and negatively affected the abundance and diversity of bacterial community, and 17 significantly decreased bacterial species in the antibiotic-treated medium, including Pseudomonas stutzeri, were identified. Further co-culture experiments revealed that P. stutzeri inhibited the growth of I. galbana, and the inhibitory activity was retained in the cell-free bacterial filtrate. These results indicated that the negative effect of bacteria was not exclusively transmitted through contact with I. galbana but could be also mediated via secretory compounds. Taken together, our findings not only fully characterized the bacterial community associated with I. galbana and how the bacterial community changed in response to antibiotic perturbations, but also provided a valuable information about the interactions between I. galbana and its associated bacteria, which might help improve the yield, and quality of I. galbana during its cultivation processes.

Leaf stage-associated resistance is correlated with phytohormones in a pathosystem-dependent manner.

It has been reported in several pathosystems that disease resistance can vary in leaves at different stages. However, how general this leaf stage-associated resistance is, and the molecular mechanism(s) underlying it, remain largely unknown. Here, we investigated the effect of leaf stage on basal resistance, effector-triggered immunity (ETI) and nonhost resistance, using eight pathosystems involving the hosts Arabidopsis thaliana, Nicotiana tabacum, and N. benthamiana and the pathogens Sclerotinia sclerotiorum, Pseudomonas syringae pv. tabaci, P. syringae pv. tomato DC3000, and Xanthomonas oryzae pv. oryzae (Xoo). We show evidence that leaf stage-associated resistance exists ubiquitously in plants, but with varying intensity at different stages in diverse pathosystems. Microarray expression profiling assays demonstrated that hundreds of genes involved in defense responses, phytohormone biosynthesis and signaling, and calcium signaling, were differentially expressed between leaves at different stages. The Arabidopsis mutants sid1, sid2-3, ein2, jar1-1, aba1 and aao3 lost leaf stage-associated resistance to S. sclerotiorum, and the mutants aba1 and sid2-3 were affected in leaf stage-associated RPS2/AvrRpt2+ -conferred ETI, whereas only the mutant sid2-3 influenced leaf stage-associated nonhost resistance to Xoo. Our results reveal that the phytohormones salicylic acid, ethylene, jasmonic acid and abscisic acid likely play an essential, but pathosystem-dependent, role in leaf stage-associated resistance.

Artificial Agrobacterium tumefaciens strains exhibit diverse mechanisms to repress Xanthomonas oryzae pv. oryzae-induced hypersensitive response and non-host resistance in Nicotiana benthamiana.

Xanthomonas oryzae pv. oryzae (Xoo) rapidly triggers a hypersensitive response (HR) and non-host resistance in its non-host plant Nicotiana benthamiana. Here, we report that Agrobacterium tumefaciens strain GV3101 blocks Xoo-induced HR in N. benthamiana when pre-infiltrated or co-infiltrated, but not when post-infiltrated at 4 h after Xoo inoculation. This suppression by A. tumefaciens is local and highly efficient to Xoo. The HR-inhibiting efficiency of A. tumefaciens is strain dependent. Strain C58C1 has almost no effect on Xoo-induced HR, whereas strains GV3101, EHA105 and LBA4404 nearly completely block HR formation. Intriguingly, these three HR-inhibiting strains employ different strategies to repress HR. Strain GV3101 displays strong antibiotic activity and thus suppresses Xoo growth. Comparison of the genotype and Xoo antibiosis activity of wild-type A. tumefaciens strain C58 and a set of C58-derived strains reveals that this Xoo antibiosis activity of A. tumefaciens is negatively, but not solely, regulated by the transferred-DNA (T-DNA) of the Ti plasmid pTiC58. Unlike GV3101, strains LBA4404 and EHA105 exhibit no significant antibiotic effect on Xoo, but rather abolish hydrogen peroxide accumulation. In addition, expression assays indicate that strains LBA4404 and EHA105 may inhibit Xoo-induced HR by suppression of the expression of Xoo type III secretion system (T3SS) effector genes hpa1 and hrpD6. Collectively, our results unveil the multiple levels of effects of A. tumefaciens on Xoo in N. benthamiana and provide insights into the molecular mechanisms underlying the bacterial antibiosis of A. tumefaciens and the non-host resistance induced by Xoo.

Genome-Wide Identification of Dicer-Like, Argonaute, and RNA-Dependent RNA Polymerase Gene Families in Brassica Species and Functional Analyses of Their Arabidopsis Homologs in Resistance to Sclerotinia sclerotiorum.

RNA silencing is an important mechanism to regulate gene expression and antiviral defense in plants. Nevertheless, RNA silencing machinery in the important oil crop Brassica napus and function in resistance to the devastating fungal pathogen Sclerotinia sclerotiorum are not well-understood. In this study, gene families of RNA silencing machinery in B. napus were identified and their role in resistance to S. sclerotiorum was revealed. Genome of the allopolyploid species B. napus possessed 8 Dicer-like (DCL), 27 Argonaute (AGO), and 16 RNA-dependent RNA polymerase (RDR) genes, which included almost all copies from its progenitor species B. rapa and B. oleracea and three extra copies of RDR5 genes, indicating that the RDR5 group in B. napus appears to have undergone further expansion through duplication during evolution. Moreover, compared with Arabidopsis, some AGO and RDR genes such as AGO1, AGO4, AGO9, and RDR5 had significantly expanded in these Brassica species. Twenty-one out of 51 DCL, AGO, and RDR genes were predicted to contain calmodulin-binding transcription activators (CAMTA)-binding site (CGCG box). S. sclerotiorum inoculation strongly induced the expression of BnCAMTA3 genes while significantly suppressed that of some CGCG-containing RNA silencing component genes, suggesting that RNA silencing machinery might be targeted by CAMTA3. Furthermore, Arabidopsis mutant analyses demonstrated that dcl4-2, ago9-1, rdr1-1, rdr6-11, and rdr6-15 mutants were more susceptible to S. sclerotiorum, while dcl1-9 was more resistant. Our results reveal the importance of RNA silencing in plant resistance to S. sclerotiorum and imply a new mechanism of CAMTA function as well as RNA silencing regulation.

Tight regulation of the interaction between Brassica napus and Sclerotinia sclerotiorum at the microRNA level.

MicroRNAs (miRNAs) are multifunctional non-coding short nucleotide molecules. Nevertheless, the role of miRNAs in the interactions between plants and necrotrophic pathogens is largely unknown. Here, we report the identification of the miRNA repertoire of the economically important oil crop oilseed rape (Brassica napus) and those involved in interacting with its most devastating necrotrophic pathogen Sclerotinia sclerotiorum. We identified 280 B. napus miRNA candidates, including 53 novel candidates and 227 canonical members or variants of known miRNA families, by high-throughput deep sequencing of small RNAs from both normal and S. sclerotiorum-inoculated leaves. Target genes of 15 novel candidates and 222 known miRNAs were further identified by sequencing of degradomes from the two types of samples. MiRNA microarray analysis revealed that 68 miRNAs were differentially expressed between S. sclerotiorum-inoculated and uninoculated leaves. A set of these miRNAs target genes involved in plant defense to S. sclerotiorum and/or other pathogens such as nucleotide binding site-leucine-rich repeat (NBS-LRR) R genes and nitric oxygen and reactive oxygen species related genes. Additionally, three miRNAs target AGO1 and AGO2, key components of post-transcriptional gene silencing (PTGS). Expression of several viral PTGS suppressors reduced resistance to S. sclerotiorum. Arabidopsis mutants of AGO1 and AGO2 exhibited reduced resistance while transgenic lines over-expressing AGO1 displayed increased resistance to S. sclerotiorum in an AGO1 expression level-dependent manner. Moreover, transient over-expression of miRNAs targeting AGO1 and AGO2 decreased resistance to S. sclerotiorum in oilseed rape. Our results demonstrate that the interactions between B. napus and S. sclerotiorum are tightly regulated at miRNA level and probably involve PTGS.

TMT-based quantitative proteomics analyses reveal novel defense mechanisms of Brassica napus against the devastating necrotrophic pathogen Sclerotinia sclerotiorum.

UNLABELLED: The white mould disease, caused by Sclerotinia sclerotiorum, is one of the most important diseases in the vital oil crop Brassica napus. Nevertheless, the defense mechanisms of B. napus against S. sclerotiorum are poorly understood. In this study, we performed comparative quantitative proteomics analyses to reveal B. napus defense mechanisms against S. sclerotiorum. The proteomes of B. napus leaves inoculated with S. sclerotiorum wild-type strain 1980 and nonpathogenic mutant strain Ep-1PB as well as empty agar plug as the control were analyzed using TMT label-based quantitative analysis technique. A total of 79, 299 and 173 proteins consistently differentially expressed between Ep-1PB- and mock-inoculated leaves, 1980- and mock-inoculated leaves, as well as 1980- and Ep-1PB-inoculated leaves, respectively, were identified. The differential expression of 12 selected proteins was confirmed by qRT-PCR analyses. The Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) and protein-protein interaction prediction analyses revealed that redox homeostasis, lipid signaling, calcium signaling, histone and DNA methylation-mediated transcription regulation and defense-related proteins such as defensin and defensin-like proteins and cyanate lyase, contribute to defense against S. sclerotiorum. Our results provide new insights into molecular mechanisms that may be involved in defense responses of B. napus to S. sclerotiorum. SIGNIFICANCE: The Sclerotinia white mould disease is one of the most important diseases in the significant oil crop Brassica napus. Nevertheless, the defense mechanisms of B. napus against S. sclerotiorum are still largely unknown to date. In this study, we addressed this issue by performing TMT label-based comparative quantitative analyses of the proteomes of B. napus leaves inoculated with S. sclerotiorum wild-type strain 1980 and nonpathogenic mutant strain Ep-1PB as well as empty agar plug as the control. Through comparative analyses on 79, 299, and 173 proteins that are consistently differentially expressed in between Ep-1PB-inoculated and the control leaves, 1980-inoculated and the control leaves, as well as 1980-inoculated and Ep-1PB-inoculated leaves, respectively, we revealed that redox homeostasis, lipid signaling, calcium signaling, histone and DNA methylation-mediated transcription regulation and defense-related proteins such as defensin and defensin-like proteins as well as cyanate lyase, contribute to B. napus defenses against S. sclerotiorum. Notably, the potential role of lipid signaling, calcium signaling, histone and DNA methylation-mediated transcription regulation and cyanate lyase in B. napus defense against S. sclerotiorum are not reported previously but rather unveiled for the first time in this study. The current study represents the most extensive analysis of the protein profile of B. napus in response to S. sclerotiorum inoculation and includes for the first time the results from comparison between plants inoculated with the wild-type strain and a nonpathogenic mutant strain of S. sclerotiorum. Collectively, our results provide new insights into the molecular mechanisms of interactions between B. napus and S. sclerotiorum.

Curcusone D, a novel ubiquitin-proteasome pathway inhibitor via ROS-induced DUB inhibition, is synergistic with bortezomib against multiple myeloma cell growth.

BACKGROUND: Ubiquitin-proteasome pathway (UPP) plays a very important role in the degradation of proteins. Finding novel UPP inhibitors is a promising strategy for treating multiple myeloma (MM). METHODS: Ub-YFP reporter assays were used as cellular UPP models. MM cell growth, apoptosis and overall death were evaluated with the MTS assay, Annexin V/PI dual-staining flow cytometry, poly (ADP-ribose) polymerase (PARP) cleavage, and PI uptake, respectively. The mechanism of UPP inhibition was analyzed by western blotting for ubiquitin, in vitro and cellular proteasomal and deubiquitinases (DUBs) activity assays. Cellular reactive oxygen species (ROS) were measured with H2DCFDA. RESULTS: Curcusone D, identified as a novel UPP inhibitor, causes cell growth inhibition and apoptosis in MM cells. Curcusone D induced the accumulation of poly-ubiquitin-conjugated proteins but could not inhibit proteasomal activity in vitro or in cells. Interestingly, the mono-ubiquitin level and the total cellular DUB activity were significantly downregulated following curcusone D treatment. Furthermore, curcusone D could induce ROS, which were closely correlated with DUB inhibition that could be nearly completely reversed by NAC. Finally, curcusone D and the proteasomal inhibitor bortezomib showed a strong synergistic effect against MM cells. CONCLUSIONS: Curcusone D is novel UPP inhibitor that acts via the ROS-induced inhibition of DUBs to produce strong growth inhibition and apoptosis of MM cells and synergize with bortezomib. GENERAL SIGNIFICANCE: The anti-MM molecular mechanism study of curcusone D will promote combination therapies with different UPP inhibitors against MM and further support the concept of oxidative stress regulating the activity of DUBs.

Fluorogenic probing of specific recognitions between sugar ligands and glycoprotein receptors on cancer cells by an economic graphene nanocomposite.

Economical nanocomposites based on π-stacking of N-acetyl glycosyl rhodamine B to graphene oxide (GO) are simply prepared. These "sweet" GO-materials are proven to be admirable for the fluorogenic recognition of specific intercellular sugar-based ligand-glycoprotein receptor interactions of interest.

Genome-wide identification and functional analyses of calmodulin genes in Solanaceous species.

BACKGROUND: Calmodulin (CaM) is a major calcium sensor in all eukaryotes. It binds calcium and modulates the activity of a wide range of downstream proteins in response to calcium signals. However, little is known about the CaM gene family in Solanaceous species, including the economically important species, tomato (Solanum lycopersicum), and the gene silencing model plant, Nicotiana benthamiana. Moreover, the potential function of CaM in plant disease resistance remains largely unclear. RESULTS: We performed genome-wide identification of CaM gene families in Solanaceous species. Employing bioinformatics approaches, multiple full-length CaM genes were identified from tomato, N. benthamiana and potato (S. tuberosum) genomes, with tomato having 6 CaM genes, N. benthamiana having 7 CaM genes, and potato having 4 CaM genes. Sequence comparison analyses showed that three tomato genes, SlCaM3/4/5, two potato genes StCaM2/3, and two sets of N. benthamiana genes, NbCaM1/2/3/4 and NbCaM5/6, encode identical CaM proteins, yet the genes contain different intron/exon organization and are located on different chromosomes. Further sequence comparisons and gene structural and phylogenetic analyses reveal that Solanaceous species gained a new group of CaM genes during evolution. These new CaM genes are unusual in that they contain three introns in contrast to only a single intron typical of known CaM genes in plants. The tomato CaM (SlCaM) genes were found to be expressed in all organs. Prediction of cis-acting elements in 5' upstream sequences and expression analyses demonstrated that SlCaM genes have potential to be highly responsive to a variety of biotic and abiotic stimuli. Additionally, silencing of SlCaM2 and SlCaM6 altered expression of a set of signaling and defense-related genes and resulted in significantly lower resistance to Tobacco rattle virus and the oomycete pathogen, Pythium aphanidermatum. CONCLUSIONS: The CaM gene families in the Solanaceous species tomato, N. benthamiana and potato were identified through a genome-wide analysis. All three plant species harbor a small set of genes that encode identical CaM proteins, which may manifest a strategy of plants to retain redundancy or enhanced quantitative gene function. In addition, Solanaceous species have evolved one new group of CaM genes during evolution. CaM genes play important roles in plant disease resistance to a variety of pathogens.