Development of an RNA Nanostructure for Effective Botrytis cinerea Control through Spray-Induced Gene Silencing without an Extra Nanocarrier.

Spray-induced gene silencing represents an eco-friendly approach for crop protection through the use of double-stranded RNA (dsRNA) to activate the RNA interference (RNAi) pathway, thereby silencing crucial genes in pathogens. The major challenges associated with dsRNA are its limited stability and poor cellular uptake, necessitating repeated applications for effective crop protection. In this study, RNA nanoparticles (NPs) were proposed as effectors in plants and pathogens by inducing the RNAi pathway and silencing gene expression. RNA structural motifs, such as hairpin-loop, kissing-loop, and tetra-U motifs, were used to link multiple siRNAs into a long, single-stranded RNA (lssRNA). The lssRNA, synthesized in Escherichia coli, self-assembled into stable RNA nanostructures via local base pairing. Comparative analyses between dsRNA and RNA NPs revealed that the latter displayed superior efficacy in inhibiting spore germination and mycelial growth of Botrytis cinerea. Moreover, RNA NPs had a more robust protective effect on plants against B. cinerea than did dsRNA. In addition, RNA squares are processed into expected siRNA in plants, thereby inhibiting the expression of the target gene. These findings suggest the potential of RNA NPs for use in plant disease control by providing a more efficient and specific alternative to dsRNA without requiring nanocarriers.

Inhibition of SARS-CoV-2 Replication by Self-Assembled siRNA Nanoparticles Targeting Multiple Highly Conserved Viral Sequences.

Coronavirus infectious disease 2019 (COVID-19), caused by severe acute respiratory virus type 2 (SARS-CoV-2), has caused a global public health crisis. As an RNA virus, the high gene mutability of SARS-CoV-2 poses significant challenges to the development of broad-spectrum vaccines and antiviral therapeutics. There remains a lack of specific therapeutics directly targeting SARS-CoV-2. With the ability to efficiently inhibit the expression of target genes in a sequence-specific way, small interfering RNA (siRNA) therapy has exhibited significant potential in antiviral and other disease treatments. In this work, we presented a highly effective self-assembled siRNA nanoparticle targeting multiple highly conserved regions of SARS-CoV-2. The siRNA sequences targeting viral conserved regions were first screened and evaluated by their thermodynamic features, off-target effects, and secondary structure toxicities. RNA motifs including siRNA sequences were then designed and self-assembled into siRNA nanoparticles. These siRNA nanoparticles demonstrated remarkable uniformity and stability and efficiently entered cells directly through cellular endocytic pathways. Moreover, these nanoparticles effectively inhibited the replication of SARS-CoV-2, exhibiting a superior inhibitory effect compared to free siRNA. These results demonstrated that these self-assembled siRNA nanoparticles targeting highly conserved regions of SARS-CoV-2 represent highly effective antiviral candidates for the treatment of infections, and are promisingly effective against current and future viral variants.

Development and application of an RNA nanostructure to induce transient RNAi in difficult transgenic plants.

The development of RNA interference (RNAi) is crucial for studying plant gene function. Its use, is limited to a few plants with well-established transgenic techniques. Spray-induced gene silencing (SIGS) introduces exogenous double-stranded RNA (dsRNA) into plants by spraying, injection, or irrigation, triggering the RNAi pathway to instantly silence target genes. As is a transient RNAi technology that does not rely on transgenic methods, SIGS has significant potential for studying gene function in plants lacking advanced transgenic technology. In this study, to enhance their stability and delivery efficiency, siRNAs were used as structural motifs to construct RNA nanoparticles (NPs) of four shapes: triangle, square, pentagon, and hexagon. These NPs, when synthesized by Escherichia coli, showed that triangular and square shapes accumulated more efficiently than pentagon and hexagon shapes. Bioassays revealed that RNA squares had the highest RNAi efficiency, followed by RNA triangles, with GFP-dsRNA showing the lowest efficiency at 4 and 7 days post-spray. We further explored the use of RNA squares in inducing transient RNAi in plants that are difficult to transform genetically. The results indicated that Panax notoginseng-derived MYB2 (PnMYB2) and Camellia oleifera-derived GUT (CoGUT) were significantly suppressed in P. notoginseng and C. oleifera, respectively, following the application of PnMYB2- and CoGUT-specific RNA squares. These findings suggest that RNA squares are highly effective in SIGS and can be utilized for gene function research in plants.

Simulation Experiment Research of Mine Roadway Simulating Test Device with Adjustable Wind Velocity and Temperature and Humidity.

The design and development process of wind velocity sensors for mining has been a challenging task due to the complexity of a large number of field tests. To resolve this problem, this study aimed to provide a comprehensive test device for the design and development of high-precision wind velocities sensor for mining. Through a combination of experiments and computational fluid dynamics (CFD), a device that can simulate the mine roadway environment was developed. The device can control the temperature, humidity, and wind velocity parameters to fully replicate the mine roadway environment. It gives designers and developers of high-precision wind velocity sensors for mining a rational and scientific testing environment. In order to quantitatively define the uniformity of air flow in the mine highway section, the research introduced the non-uniformity determination method. The approach was expanded to assess the cross-sectional uniformity of temperature and humidity. The wind velocity within the machine can increase to 8.5 m/s by selecting the right kind of fan. The minimum wind velocity non-uniformity at this moment is 2.30%. The device's internal temperature can be raised to 38.23 °C and its humidity level can be increased to 95.09% by carefully crafting the rectifier orifice plate's structure. At this time, the lowest temperature non-uniformity is 2.22%, and the lowest humidity non-uniformity is 2.40%. The device's average wind velocity is 4.37 m/s, its average temperature is 37.7 °C, as well as its average humidity is 95%, per the emulate results. The device's non-uniformity in wind velocity, temperature, and humidity is 2.89%, 1.34%, and 2.23%, respectively. It is capable of simulating the mine roadway environment in its entirety.

Genome-wide identification and validation of tomato-encoded sRNA as the cross-species antifungal factors targeting the virulence genes of Botrytis cinerea.

Recent evidence shows that small RNAs are transferred from a species to another through cross-species transmission and exhibit biological activities in the receptor. In this study, we focused on tomato-derived sRNAs play a role of defense against Botrytis cinerea. Bioinformatics method was firstly employed to identify tomato-encoded sRNAs as the cross-species antifungal factors targeting B. cinerea genes. Then the expression levels of some identifed sRNAs were checked in B. cinerea-infected plant using qRT-PCR method. Exogenic RNA-induced gene silences analysis were performed to investigate the antifungal roles of the sRNAs, and the target genes in B. cinerea of antifungal sRNAs would be confirmed by using co-expression analysis. Results showed that a total of 21 B.cinerea-induced sRNAs with high abundance were identified as the cross-kingdom regulator candidates. Among them, three sRNAs containing a miRNA (miR396a-5p) and two siRNA (siR3 and siR14) were selected for experimental validation and bioassay analysis. qRT-PCR confirmed that all of these 3 sRNAs were induced in tomato leaves by B. cinerea infection. Correspondingly, 4 virulence genes of B. cinerea respectively targeted by these 3 sRNAs were down-regulated. Bioassay revealed that all of these 3 cross-species sRNAs could inhibit the virulence and spore gemination of B. cinerea. Correspondingly, the coding genes of B. cinerea targeted by these sRNAs were also down-regulated. Moreover, the virulence inhibition by double strand sRNA was more effective than that by single strand sRNA. The inhibition efficiency of sRNA against B. cinerea increased with the increase of its concentration. Our findings provide new evidence into the coevolution of pathogens and host plants, as well as new directions for the use of plant-derived sRNAs to control pathogens.

Analysis of miRNAs responsive to long-term calcium deficiency in tef (Eragrostis tef (Zucc.) Trotter).

MicroRNAs (miRNAs) play an important role in growth, development, stress resilience, and epigenetic modifications of plants. However, the effect of calcium (Ca2+) deficiency on miRNA expression in the orphan crop tef (Eragrostis tef) remains unknown. In this study, we analyzed expression of miRNAs in roots and shoots of tef in response to Ca2+ treatment. miRNA-seq followed by bioinformatic analysis allowed us to identify a large number of small RNAs (sRNAs) ranging from 17 to 35 nt in length. A total of 1380 miRNAs were identified in tef experiencing long-term Ca2+ deficiency while 1495 miRNAs were detected in control plants. Among the miRNAs identified in this study, 161 miRNAs were similar with those previously characterized in other plant species and 348 miRNAs were novel, while the remaining miRNAs were uncharacterized. Putative target genes and their functions were predicted for all the known and novel miRNAs that we identified. Based on gene ontology (GO) analysis, the predicted target genes are known to have various biological and molecular functions including calcium uptake and transport. Pairwise comparison of differentially expressed miRNAs revealed that some miRNAs were specifically enriched in roots or shoots of low Ca2+-treated plants. Further characterization of the miRNAs and their targets identified in this study may help in understanding Ca2+ deficiency responses in tef and related orphan crops.

Molecular mechanism of modulating miR482b level in tomato with botrytis cinerea infection.

BACKGROUND: Plant miRNAs are involved in the response to biotic and abiotic stresses by altering their expression levels, and they play an important role in the regulation of plant resistance to stress. However, the molecular mechanism that regulates the expression levels of miRNAs in plants with biotic and abiotic stress still needs to be explored. Previously, we found that the expression of the miR482 family was changed in tomato infected by Botrytis cinerea. In this study, we investigated and uncovered the mechanism underlying the response of miR482 to B. cinerea infection in tomato. RESULTS: First, RT-qPCR was employed to detect the expression patterns of miR482b in tomato infected by B. cinerea, and results showed that miR482b primary transcripts (pri-miR482b) were up-regulated in B. cinerea-infected leaves, but the mature miR482b was down-regulated. Subsequently, we used rapid amplification cDNA end method to amplify the full-length of pri-miR482b. Result showed that the pri-miR482b had two isoforms, with the longer one (consisting 300 bp) having an extra fragment of 53 bp in the 3'-end compared with the shorter one. In vitro Dicer assay indicated that the longer isoform pri-miR482b-x1 had higher efficiency in the post-transcriptional splicing of miRNA than the shorter isoform pri-miR482b-x2. In addition, the transcription level of mature miR482b was much higher in transgenic Arabidopsis overexpressing pri-miR482b-x1 than that in OE pri-miR482b-x2 Arabidopsis. These results confirmed that this extra 53 bp in pri-miR482b-x1 might play a key role in the miR482b biogenesis of post-transcription processing. CONCLUSIONS: Extra 53 bp in pri-miR482b-x1 enhanced miR482b biogenesis, which elevated the transcription level of miR482b. This study clarified the response of miR482 to B. cinerea infection in tomato, thereby helping us further understand the molecular mechanisms that regulate the expression levels of other miRNAs.

Characterization of NAC family genes in Salvia miltiorrhiza and NAC2 potentially involved in the biosynthesis of tanshinones.

The NAC (NAM, ATAF, and CUC) family members are specific transcription factors in plants. The large family is involved in many plant growth and developmental processes, as well as in abiotic/biotic stress responses. It has been well studied in the genomes of various plants, including Arabidopsis thaliana, tomato, and quinoa. However, identification and functional studies of NAC family members in medicinal Salvia miltiorrhiza are limited. Here, we systematically identified 84 NAC genes and named them according to their gene IDs in the recently sequenced genome. The phylogeny of NAC family protein sequences was analyzed using bioinformatics methods, which divided them into nine subfamilies. Then, their chromosomal locations, gene structures and conserved domains were analyzed comprehensively. To further investigate the regulatory functions of NACs in S. miltiorrhiza, we analyzed the response of 10 selected NAC genes to methyl jasmonate and used NAC2 for transgenic experiments. The overexpression of Sm-NAC2 decreased the tanshinone I and IIA contents by 56% and 62%, respectively. However, Sm-NAC2-RNAi promoted the accumulation of four tanshinones, tanshinone I, tanshinone IIA, cryptotanshinone, and dihydrotanshinone I, which increased 3.68-, 4.1-, 3.13- and 5.9- fold, respectively, compared with wild type. In the tanshinone biosynthetic pathways, the overexpression of Sm-NAC2 down-regulated CYP76AH1, and the silencing of Sm-NAC2 up-regulated the expression levels of HMGR1, DXS2, KSL2, and CYP76AH1. This study provides information on the evolution of Sm-NAC genes and their possible functions, and it lays a foundation for further research into the NAC family-associated regulation of tanshinone biosynthesis.

miR319c acts as a positive regulator of tomato against Botrytis cinerea infection by targeting TCP29.

miR319 family is one of the oldest and most conservative miRNA families in plant and plays an important role in plant development and abiotic stress response. In our previous study, the abundance of sly-miR319c was increased in tomatoes infected by B. cinerea, but the roles and regulatory mechanisms of sly-miR319c in B. cinerea-infected tomato remain unclear. In this study, we confirmed that miR319c was increased in tomato with B. cinerea infection. In contrast, A TCP transcript factor, TCP29, targeted by sly-miR319c was decreased in B. cinerea-infected tomato. Therefore, transgenic Arabidopsis overexpressing sly-miR319c or its target were generated for understanding the biological roles and molecular mechanism of miR319c in B.cinerea-infected plants. Results showed that miR319c overexpression improved the resistance of transgenic plants to B. cinerea, whereas TCP29 overexpression increased the susceptibility of transgenic plant to B. cinerea. So far, TCP transcription factors have been reported mainly in developmental processes. Our data indicate that TCP29 act as a negative regulator to B.cinerea infection. In conclusion, our results indicate that sly-miR319c is a positive regulator of tomato resistance to B. cinerea infection by targeting TCP29.

Novel tomato miRNA miR1001 initiates cross-species regulation to suppress the conidiospore germination and infection virulence of Botrytis cinerea in vitro.

Recent evidence has shown that microRNAs are transferred from one species to another through cross-species transmission and exhibit biological activities in the receptor. However, the cross-kingdom regulation of pathogen virulence by plant-derived miRNAs is rarely reported. This study investigated the regulatory role of novel tomato miRNA miR1001 in the growth and development of Botrytis cinerea. Results showed that miR1001 inhibited the virulence of B. cinerea-infected plants, and the inhibitory effect of miR1001/miR1001* was stronger than that of miR1001. Moreover, miR1001 exerted a significant inhibitory effect on the conidiospore germination of B. cinerea. Degradome-seq experiment showed that miR1001 can directly target the Bcin03g02170.1 and Bcin10g01400.1 genes, which respectively encode the ATP-dependent metallopeptidase and cysteine-type endopeptidase, in B. cinerea. The interactions of both targets with miR1001 were further confirmed by using transient co-expression in tobacco. Real-time RT-PCR analysis showed that the expression levels of the two target genes were significantly downregulated in B. cinerea with miR1001 treatment. Our findings provide new evidence into the coevolution of pathogens and host plants, as well as new directions for the use of plant-derived miRNAs to control pathogens.

[Cloning,subcellular localization and spatio-temporal expression analysis of a flavonoid 3-O-glucosyltransferase gene( SmUF3GT) in Salvia miltiorrhiza].

The family of flavonoid 3-O-glucosyltransferase catalyzes the modification of anthocyanin from unstable-structure to stable-structure. In this study,based on homology cloning and transcriptome library,we isolated the full-length c DNA of UDP-glucose: flavonoid 3-O-glucosyltransferase( named SmUF3GT) from the flower tissues of S. miltiorrhiza. This gene was consisted of 1 353 bp open reading frames( ORF) encoding 450 amino acids. And the SmUF3GT protein was performed for the bioinformatic analysis. Our results showed that the protein was preliminary localized in the Golgi and peroxisome of cytosol,as well as plasma membrane and cell nuclear.QRT-PCR analyses indicated that SmUF3GT expressed differently in all tissues and organs but roots of S. miltiorrhiza and S. miltiorrhiza f.alba. During floral development,the expression of SmUF3GT showed a trend of rising fist and then down in purple-flower Danshen,whereas decreasing sharply fist and then slowly in white-flower Danshen. The present study provides basic information for further research on the network of synthesis and accumulation of flavonoids in S.miltiorrhiza.

miR394 Acts as a Negative Regulator of Arabidopsis Resistance to B. cinerea Infection by Targeting LCR.

Gray mold of tomato is caused by the pathogen Botrytis cinerea. MicroRNAs play a crucial role in the biotic and abiotic stress responses of plants and regulate their targets by gene silencing. miR394 is an ancient and conserved miRNA in plants, and it participates in the regulation of plant development and stress responses. In our previous study, miR394 was found to respond to B. cinerea infection in tomato, but the roles and regulatory mechanisms of miR394 in B. cinerea-infected tomato remain unclear. miR394 was down-regulated in tomato in response to B. cinerea infection, showing an expression pattern opposite to the previous finding that miR394 was up-regulated in tomato cv. Jinpeng 1 infected by B. cinerea. We obtained transgenic Arabidopsis overexpressing miR394, which resulted in low expression levels of its target LEAF CURLING RESPONSIVENESS (LCR). Leaf lesion size and trypan blue staining showed that miR394 overexpression led to increased sensitivity of transgenic Arabidopsis to B. cinerea compared to wild type. We also detected changes in the expression levels of stress-related miRNAs, including miR159, miR156, miR168, and miR172. In the transgenic plants, it indicated potential cross talk between these miRNAs and miR394, except for miR159. miR394 also enhanced the expression of ARGONAUTE 1 (AGO1), DSRNA-BINDING PROTEIN 4 (DRB4) and the RNA-binding protein gene DAWDLE (DDL), which are involved in the pathways of miRNA biosynthesis and regulation, suggesting that miR394 overexpression has a feedback effect on these genes. Our data indicate that overexpression of miR394 in Arabidopsis increased the susceptibility of plants to B. cinerea by affecting the expression of its target gene LCR along with a number of key genes involved in plant miRNA metabolism (AGO1). Thus, miR394 is a negative regulator of Arabidopsis resistance to B. cinerea infection by targeting LCR.

[Cloning and expression analysis of protein kinase SmSnRK2.4 from Salvia miltiorrhiza].

Sucrose non-fermenting 1-related protein kinase 2(SnRK2) plays a key role in abiotic stress signaling in plants. In this study, we cloned a SmSnRK2.4 gene belonging to subclass I of SnRK2 from Salvia miltiorrhiza by screening its transcriptome database. The SmSnRK2.4 gene contains 8 introns and 9 exons, with a 1 068 bp open reading frame encoding a polypeptide of 355 amino acids, the predicted molecular mass of which is 40.63 kDa. Prokaryotic expression of SmSnRK2.4 protein using pMAL-c2X as the expression vector displayed that the recombinant protein of SmSnRK2.4 gene in E. coli was consistent with the predicted size. A 3 000 bp promoter sequence of SmSnRK2.4 contained some stress-responsive elements and hormone-responsive elements. Quantitative real-time PCR analysis revealed that the expression of SmSnRK2.4 in root was much higher than that in stem and leaf, SmSnRK2.4 was strongly induced by PEG stress, weakly induced by ABA stress. This research provided a basis for further study of the SmSnRK2.4 gene playing the role in accumulate mechanism of secondary metabolites in S. miltiorrhiza under drought.

Genome-wide identification and characterization of phased small interfering RNA genes in response to Botrytis cinerea infection in Solanum lycopersicum.

Phased small interfering RNAs (phasiRNAs) are encoded by a novel class of genes known as phasiRNA producing (PHAS) genes. These genes play important regulatory roles by targeting protein coding transcripts in plant species. In this study, 91 regions were identified as potential PHAS loci in tomato, with additional evidence that seven of them can be triggered by five miRNAs. Among the identified loci, 51 were located in genic regions, and the remaining 40 were located in intergenic regions. The transient overexpression of PHAS15 and PHAS26 demonstrated that phasiRNAs predicted by PhaseTank were indeed generated from their respective PHAS loci. Using sRNA-seq data from B. cinerea-infected tomato leaves, we identified 50 B. cinerea-responsive phasiRNAs with increased abundance and five with decreased abundance. Moreover, 164 targets of these differentially expressed phasiRNAs were predicted, and 94 of them were confirmed experimentally using degradome data. Gene ontology analysis of the targets revealed an enrichment of genes with functions related to defense responses and signaling regulation. These results suggest that a large number of endogenous siRNAs, such as phasiRNAs, have not yet been identified in tomato and underscore the urgent need to systematically identify and functionally analyze siRNAs in tomato.

Comparative transcriptome analysis of the different tissues between the cultivated and wild tomato.

Although domesticated tomato is cultivated by wild tomato, there are a lot of differences between cultivated tomato and wild tomato, such as shape, physiological function and life history. Many studies show that wild tomato has better salt resistance and drought resistance. In addition to, domesticated tomato's fruit is bigger and has more nutritious than wild tomato. The different features are closely related to differentially expressed genes. We identified 126 up-regulated differentially expressed genes and 87 down-regulated differentially expressed genes in cultivated tomato and wild tomato by RNA-Seq. These differentially expressed genes may be associated with salt resistance, drought resistance and fruit nutrition. These differentially expressed genes also further highlight the large-scale reconstruction between wild and cultivated species. In this paper, we mainly study GO enrichment analysis and pathway analysis of the differentially expressed genes. After GO and pathway enrichment analysis, a set of significantly enriched GO annotations and pathways were identified for the differentially expressed genes. What's more, we also identified long non-coding RNAs and mRNAs in the two species and analyzed its essential features. In addition to, we construct a co-expression network of long non-coding RNAs and mRNAs, and annotate mRNAs associated with long non-coding RNAs as target genes, and speculate the regulation function of long non-coding RNAs. In total, our results reveal the effects of artificial and natural selection on tomato's transcript, providing scientific basis for tomato's research in the future.

Identification and Characterization of Salvia miltiorrhizain miRNAs in Response to Replanting Disease.

Replanting disease is a major factor limiting the artificial cultivation of the traditional Chinese medicinal herb Salvia miltiorrhiza. At present, little information is available regarding the role of miRNAs in response to replanting disease. In this study, two small RNA libraries obtained from first-year (FPR) and second-year plant (SPR) roots were subjected to a high-throughput sequencing method. Bioinformatics analysis revealed that 110 known and 7 novel miRNAs were annotated in the roots of S. miltiorrhiza. Moreover, 39 known and 2 novel miRNAs were identified and validated for differential expression in FPR compared with SPR. Thirty-one of these miRNAs were further analyzed by qRT-PCR, which revealed that 5 miRNAs negatively regulated the expression levels of 7 target genes involved in root development or stress responses. This study not only provides novel insights into the miRNA content of S. miltiorrhiza in response to replanting disease but also demonstrates that 5 miRNAs may be involved in these responses. Interactions among the differentially expressed miRNAs with their targets may form an important component of the molecular basis of replanting disease in S. miltiorrhiza.

DsTRD: Danshen Transcriptional Resource Database.

Salvia miltiorrhiza has been comprehensively studied as a medicinal model plant. However, research progress on this species is significantly hindered by its unavailable genome sequences and limited number of expressed sequence tags in the National Center for Biotechnology Information database. Thus, a transcript database must be developed to assist researchers to browse, search, and align sequences for gene cloning and functional analysis in S. miltiorrhiza. In this study, the Danshen Transcriptional Resource Database (DsTRD) was built using 76,531 transcribed sequences assembled from 12 RNA-Seq transcriptomes. Among these 12 RNA-seq data, ten were downloaded from NCBI database. The remaining two were enced on the Hiseq2000 platform using the stem and hairy-root of S. miltiorrhiza. The transcripts were annotated as protein-coding RNAs, long non-coding RNAs, microRNA precursors, and phased secondary small-interfering RNA genes through several bioinformatics methods. The tissue expression levels for each transcript were also calculated and presented in terms of RNA-Seq data. Overall, DsTRD facilitates browsing and searching for sequences and functional annotations of S. miltiorrhiza. DsTRD is freely available at http://bi.sky.zstu.edu.cn/DsTRD/home.php.

Proteome-Wide Identification of Lysine Succinylation in the Proteins of Tomato (Solanum lycopersicum).

Post-translational modification of proteins through lysine succinylation plays important regulatory roles in living cells. Lysine succinylation was recently identified as a novel post-translational modification in Escherichia coli, yeast, Toxoplasma gondii, HeLa cells, and mouse liver. Interestingly, only a few sites of lysine succinylation have been detected in plants to date. In this study, we identified 347 sites of lysine succinylation in 202 proteins in tomato by using high-resolution mass spectrometry. Succinylated proteins are implicated in the regulation of diverse metabolic processes, including chloroplast and mitochondrial metabolism. Bioinformatic analysis showed that succinylated proteins are evolutionarily conserved and involved in various cellular functions such as metabolism and epigenetic regulation. Moreover, succinylated proteins exhibit diverse subcellular localizations. We also defined six types of definitively conserved succinylation motifs. These results provide the first in-depth analysis of the lysine succinylome and novel insights into the role of succinylation in tomato, thereby elucidating lysine succinylation in the context of cellular physiology and metabolite biosynthesis in plants.

The tRNA-Derived Small RNAs Regulate Gene Expression through Triggering Sequence-Specific Degradation of Target Transcripts in the Oomycete Pathogen Phytophthora sojae.

Along with the well-studied microRNA (miRNA) and small interfering RNA (siRNA) is a new class of transfer RNA-derived small RNA (tsRNA), which has recently been detected in multiple organisms and is implicated in gene regulation. However, while miRNAs and siRNAs are known to repress gene expression through sequence-specific RNA cleavage or translational repression, how tsRNAs regulate gene expression remains unclear. Here we report the identification and functional characterization of tsRNAs in the oomycete pathogen Phytophthora sojae. We show that multiple tRNAs are processed into abundant tsRNAs, which accumulate in a similar developmental stage-specific manner and are negatively correlated with the expression of predicted target genes. Degradome sequencing and 5' RLM RACE experiments indicate tsRNAs can trigger degradation of target transcripts. Transient expression assays using GUS sensor constructs confirmed the requirement of sequence complementarity in tsRNA-mediated RNA degradation in P. sojae. Our results show that the tsRNA are a class of functional endogenous sRNAs and suggest that tsRNA regulate gene expression through inducing sequence-specific degradation of target RNAs in oomycetes.

Identification and characterization of cucumber microRNAs in response to Pseudoperonospora cubensis infection.

MicroRNAs (miRNAs) regulate the expression of genes related to several stress responses, including fungal infection, in plants. However, the miRNA-mediated gene regulatory networks in cucumbers that respond to Pseudoperonospora cubensis stress remain unexplored. In this study, the miRNA expression patterns in response to P. cubensis stress in cucumbers were investigated through high-throughput sequencing. A total of 123 known miRNAs and 4 novel miRNAs were identified, and their corresponding expressions were detected in mock- and P. cubensis-inoculated leaves. Three novel and 39 known miRNAs were found to be differentially expressed in P. cubensis-infected leaves. The results of 5'-RLM-RACE confirmed that miR164b, miR156h, miR171e, miR160b, and miR159f targeted No Apical Meristem domain protein, squamosa promoter binding protein-like class transcription factor, GRAS family transcription factor, Auxin response factor ARF16, and a conserved gene of unknown function, respectively. The expression patterns of these miRNAs were also determined through quantitative reverse transcription polymerase chain reaction (qRT-PCR). All of these miRNAs, except for miR156h, can respond to P. cubensis infection in cucumber leaves. In addition, the results of qRT-PCR revealed that the targets negatively correlated with their corresponding miRNAs (miR164b, miR171e, miR160b, and miR159f).