WIPK-NtLTP4 pathway confers resistance to Ralstonia solanacearum in tobacco.

KEY MESSAGE: WIPK-NtLTP4 module improves the resistance to R. solanacearum via upregulating the expression of defense-related genes, increasing the antioxidant enzyme activity, and promoting stomatal closure in tobacco. Lipid transfer proteins (LTPs) are a class of small lipid binding proteins that play important roles in biotic and abiotic stresses. The previous study revealed that NtLTP4 positively regulates salt and drought stresses in Nicotiana tabacum. However, the role of NtLTP4 in biotic stress, especially regarding its function in disease resistance remains unclear. Here, the critical role of NtLTP4 in regulating resistance to Ralstonia solanacearum (R. solanacearum), a causal agent of bacterial wilt disease in tobacco, was reported. The NtLTP4-overexpressing lines markedly improved the resistance to R. solanacearum by upregulating the expression of defense-related genes, increasing the antioxidant enzyme activity, and promoting stomatal closure. Moreover, NtLTP4 interacted with wound-induced protein kinase (WIPK; a homolog of MAPK3 in tobacco) and acted in a genetically epistatic manner to WIPK in planta. WIPK could directly phosphorylate NtLTP4 to positively regulate its protein abundance. Taken together, these results broaden the knowledge about the functions of the WIPK-NtLTP4 module in disease resistance and may provide valuable information for improving tobacco plant tolerance to R. solanacearum.

Ultrahigh-activity immune inducer from Endophytic Fungi induces tobacco resistance to virus by SA pathway and RNA silencing.

BACKGROUND: Plant viruses cause severe economic losses in agricultural production. An ultrahigh activity plant immune inducer (i.e., ZhiNengCong, ZNC) was extracted from endophytic fungi, and it could promote plant growth and enhance resistance to bacteria. However, the antiviral function has not been studied. Our study aims to evaluate the antiviral molecular mechanisms of ZNC in tobacco. RESULTS: Here, we used Potato X virus (PVX), wild-type tobacco and NahG transgenic tobacco as materials to study the resistance of ZNC to virus. ZNC exhibited a high activity in enhancing resistance to viruses and showed optimal use concentration at 100-150 ng/mL. ZNC also induced reactive oxygen species accumulation, increased salicylic acid (SA) content by upregulating the expression of phenylalanine ammonia lyase (PAL) gene and activated SA signaling pathway. We generated transcriptome profiles from ZNC-treated seedlings using RNA sequencing. The first GO term in biological process was positive regulation of post-transcriptional gene silencing, and the subsequent results showed that ZNC promoted RNA silencing. ZNC-sprayed wild-type leaves showed decreased infection areas, whereas ZNC failed to induce a protective effect against PVX in NahG leaves. CONCLUSION: All results indicate that ZNC is an ultrahigh-activity immune inducer, and it could enhance tobacco resistance to PVX at low concentration by positively regulating the RNA silencing via SA pathway. The antiviral mechanism of ZNC was first revealed in this study, and this study provides a new antiviral bioagent.

Global transcriptome analyses reveal the molecular signatures in the early response of potato (Solanum tuberosum L.) to Phytophthora infestans, Ralstonia solanacearum, and Potato virus Y infection.

MAIN CONCLUSION: Specific and common genes including transcription factors, resistance genes and pathways were significantly induced in potato by Phytophthora infestans, Ralstonia solanacearum, and Potato virus Y infection. The three major pathogens, namely, Phytophthora infestans, Ralstonia solanacearum, and Potato virus Y, can cause late blight, bacterial wilt, and necrotic ringspot, respectively, and thus severely reduce the yield and quality of potatoes (Solanum tuberosum L.). This study was the first to systematically analyze the relationship between transcriptome alterations in potato infected by these pathogens at the early stages. A total of 75,500 unigenes were identified, and 44,008 were annotated into 5 databases, namely, non-redundant (NR), Swiss-Prot protein, clusters of orthologous groups for eukaryotic complete genomes (KOG), Gene Ontology (GO), and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases. A total of 6945 resistance genes and 11,878 transcription factors (TFs) were identified from all transcriptome data. Differential expression analysis revealed that 13,032 (9490 specifics), 9877 (6423 specifics), and 6661 (4144 specifics) differentially expressed genes (DEGs) were generated from comparisons of the P. infestans/control (Pi vs. Pi-CK), R. solanacearum/control (Rs vs. Rs-CK), and PVY/control (PVY vs. PVY-CK) treatments, respectively. The specific DEGs from the 3 comparisons were assigned to 13 common pathways, such as biosynthesis of amino acids, plant hormone signal transduction, carbon metabolism, and starch and sucrose metabolism. Weighted Gene Co-Expression Network Analysis (WGCNA) identified many hub unigenes, of which several unigenes were reported to regulate plant immune responses, such as FLAGELLIN-SENSITIVE 2 and chitinases. The present study provide crucial systems-level insights into the relationship between transcriptome changes in potato infected with the three pathogens. Moreover, this study presents a theoretical basis for breeding broad-spectrum and specific pathogen-resistant cultivars.

High-degree and broad-spectrum resistance mediated by a combination of NIb siRNA and miRNA suppresses replication of necrotic and common strains of potato virus Y.

In plants, viral replication can be inhibited through gene silencing, which is mediated by short interfering RNA (siRNA) or microRNA (miRNA). However, under natural conditions, viruses are extremely susceptible to mutations that may decrease the efficiency of cleavage of these small RNAs (sRNAs). Therefore, a single sRNA may not provide a sufficient degree of viral resistance to transgenic plants. Potato virus Y necrotic strain (PVYN) and Potato virus Y common strain (PVYO) are the two major PVY strains that cause systemic necrosis and mottling, respectively, in tobacco. In this study, we designed specific siRNAs and miRNAs to target two regions of the PVYO replicase gene (NIb). Eight plant expression vectors containing one or two sRNAs were constructed. Luciferase activity assays showed that the designed sRNAs successfully cleaved the NIb gene of PVYO and PVYN, and the vector carrying a combined siRNA- and miRNA-based short hairpin RNA (shRNA) demonstrated the strongest inhibitory effect. These effects were confirmed through the acquisition of PVYO and PVYN resistance in transgenic sRNA-expressing Nicotiana tabacum plants. This phenomenon could be related to a plant defense mechanism in which siRNA and miRNA pathways are complementary and interact to achieve gene silencing. Furthermore, there is a tendency for the homologous small RNA sequences (PVYO) to be more effective in conferring resistance than those with imperfect homology (PVYN). Overall, these findings confirm that the use of a combined siRNA- and miRNA-based shRNAs is a promising approach for introducing viral resistance to plants through genetic engineering.

Silencing suppressors of rice black-streaked dwarf virus and rice stripe virus hijack the 26S proteasome of Laodelphax striatellus to facilitate virus accumulation and transmission.

BACKGROUND: Rice black-streaked dwarf virus (RBSDV) is transmitted by small brown planthopper (Laodelphax striatellus [L. striatellus]) and causes devastating disease in rice. P9-1 has silencing suppression activity and is the key protein for viroplasm formation in RBSDV-infected plants and insects; however, its exact function is poorly understood. RESULTS: In this study, the P9-1 of RBSDV interacted with L. striatellus 26S proteasome subunit RPN8. RBSDV accumulation in L. striatellus increased after the 26S proteasome was disrupted by silencing the RPN8 expression. This finding indicated that L. striatellus 26S proteasome played a defense role against RBSDV infection by regulating RBSDV accumulation. Further investigations revealed that P9-1 could competitively bind to RPN8 with RPN7, thereby disrupting the assembly of 26S proteasome in L. striatellus and promoting the infection of RBSDV in insect vectors, and further affecting the transmission of the virus to rice by insect vectors. Similar to P9-1, rice stripe virus (RSV) NS2, a weak silencing suppressor, regulated virus accumulation and transmission by hijacking RPN8 to interfere with the function of 26S proteasome in L. striatellus. CONCLUSION: These results suggest that viruses promote their own infection via interfering with ubiquitination pathway of insect vectors, and this mechanism might be of universal importance. These findings provide a new insight into the mechanism of virus transmission in insect vectors. © 2022 Society of Chemical Industry.

Production of transgenic rice new germplasm with strong resistance against two isolations of Rice stripe virus by RNA interference.

Rice stripe disease, with the pathogen Rice stripe virus (RSV), is one of the most widespread and severe virus diseases. Cultivating a resistant breed is an essential and efficient method in preventing rice stripe disease. Following RNA interference (RNAi) theory, we constructed three RNAi binary vectors based on coat protein (CP), special-disease protein (SP) and chimeric CP/SP gene sequence. Transgenic lines of rice cv. Yujing6 were generated through Agrobacterium-mediated transformation. We inoculated T1 generation plants from each line derived from CP/SP, CP, and SP transgenic rice plants with two RSV isolates from Shandong Province and Jiangsu Province using viruliferous vector insects. In these resistance assays, chimeric CP/SP RNAi lines showed stronger resistance against two isolates than CP or SP single RNAi lines. Stable integration and expression of RNAi transgenes were confirmed by Southern and northern blot analysis of independent transgenic lines. In the resistant transgenic lines, lower levels of transgene transcripts and specific short interference RNAs were observed relative to the susceptible transgenic plant, which showed that virus resistance was increased by RNAi. Genetic analysis demonstrated that transgene and virus resistance was stably inherited in the T2 progeny plants.

A nonspecific lipid transfer protein, StLTP10, mediates resistance to Phytophthora infestans in potato.

Nonspecific lipidtransfer proteins (nsLTPs), which are small, cysteine-rich proteins, belong to the pathogenesis-related protein family, and several of them act as positive regulators during plant disease resistance. However, the underlying molecular mechanisms of these proteins in plant immune responses are unclear. In this study, a typical nsLTP gene, StLTP10, was identified and functionally analysed in potato. StLTP10 expression was significantly induced by Phytophthora infestans, which causes late blight in potato, and defence-related phytohormones, including abscisic acid (ABA), salicylic acid, and jasmonic acid. Characterization of StLTP10-overexpressing and knockdown lines indicated that StLTP10 positively regulates plant resistance to P. infestans. This resistance was coupled with enhanced expression of reactive oxygen species scavenging- and defence-related genes. Furthermore, we identified that StLTP10 physically interacts with ABA receptor PYL4 and affects its subcellular localization. These two proteins work together to regulate stomatal closure during pathogen infection. Interestingly, we also found that wound-induced protein kinase interacts with StLTP10 and positively regulates its protein abundance. Taken together, our results provide insight into the role of StLTP10 in resistance to P. infestans and suggest candidates to enhance broad-spectrum resistance to pathogens in potato.

Production of double-stranded RNA for interference with TMV infection utilizing a bacterial prokaryotic expression system.

In many species, the introduction of double-stranded RNA (dsRNA) induces potent and specific gene silencing, a phenomenon called RNA interference (RNAi). RNAi is the process of sequence-specific, posttranscriptional gene silencing (PTGS) in animals and plants, mediated by dsRNA homologous to the silenced genes. In plants, PTGS is part of a defense mechanism against virus infection, and dsRNA is the pivotal factor that induces gene silencing. Here, we report an efficient method that can produce dsRNA using a bacterial prokaryotic expression system. Using the bacteriophage lambda-dependent Red recombination system, we knocked out the rnc genes of two different Escherichia coli strains and constructed three different vectors that could produce dsRNAs. This work explores the best vector/host combinations for high output of dsRNA. In the end, we found that strain M-JM109 or the M-JM109lacY mutant strain and the vector pGEM-CP480 are the best choices for producing great quantities of dsRNA. Resistance analyses and Northern blot showed that Tobacco mosaic virus infection could be inhibited by dsRNA, and the resistance was an RNA-mediated virus resistance. Our findings indicate that exogenous dsRNA could form the basis for an effective and environmentally friendly biotechnological tool that protects plants from virus infections.

Comprehensive transcriptomics and proteomics analyses of rice stripe virus-resistant transgenic rice.

Stable transgenic rice line (named KRSV-1) with strong resistance against rice stripe virus was generated using the gene sequence of disease-specific protein by RNA interference. Comprehensive safety assessment of transgenic plants has turned into a significant field of genetic modification food safety. In this study, a safety assessment of KRSV-1 was carried out in a stepwise approach. The molecular analysis exhibited that KRSV-1 harbored one copy number of transgene, which was integrated into the intergenic non-coding region of chromosome 2 associated with inter-chromosomal translocations of 1.6-kb segments of chromosome 8. Then, transcriptomics and proteomics analyses were carried out to detect the unintended effects as a result of the integration of the transgene. Although 650 dramatically differentially expressed genes (DDEGs) and 357 differentially expressed proteins were detected between KRSV-1 and wild-type (WT) by transcriptomics and proteomics analyses, no harmful members in the form of toxic proteins and allergens were observed. Encouragingly, the nutritional compositions of seeds from KRSV-1 were comparable with WT seeds. The results of this entire study of molecular analysis, transcriptome and proteome profile of KRSV-1 revealed that no detrimental changes in the form of toxic proteins and allergens were detected in the transgenic rice line due to the integration of the transgene.

NtLTP4, a lipid transfer protein that enhances salt and drought stresses tolerance in Nicotiana tabacum.

Lipid transfer proteins (LTPs), a class of small, ubiquitous proteins, play critical roles in various environmental stresses. However, their precise biological functions remain unknown. Here we isolated an extracellular matrix-localised LTP, NtLTP4, from Nicotiana tabacum. The overexpression of NtLTP4 in N. tabacum enhanced resistance to salt and drought stresses. Upon exposure to high salinity, NtLTP4-overexpressing lines (OE lines) accumulated low Na+ levels. Salt-responsive genes, including Na+/H+ exchangers (NHX1) and high-affinity K+ transporter1 (HKT1), were dramatically higher in OE lines than in wild-type lines. NtLTP4 might regulate transcription levels of NHX1 and HKT1 to alleviate the toxicity of Na+. Interestingly, OE lines enhanced the tolerance of N. tabacum to drought stress by reducing the transpiration rate. Moreover, NtLTP4 could increase reactive oxygen species (ROS)-scavenging enzyme activity and expression levels to scavenge excess ROS under drought and high salinity conditions. We used a two-hybrid yeast system and screened seven putative proteins that interact with NtLTP4 in tobacco. An MAPK member, wound-induced protein kinase, was confirmed to interact with NtLTP4 via co-immunoprecipitation and a firefly luciferase complementation imaging assay. Taken together, this is the first functional analysis of NtLTP4, and proves that NtLTP4 positively regulates salt and drought stresses in N. tabacum.

GhWRKY25, a group I WRKY gene from cotton, confers differential tolerance to abiotic and biotic stresses in transgenic Nicotiana benthamiana.

WRKY transcription factors are involved in various processes, ranging from plant growth to abiotic and biotic stress responses. Group I WRKY members have been rarely reported compared with group II or III members, particularly in cotton (Gossypium hirsutum). In this study, a group I WRKY gene, namely, GhWRKY25, was cloned from cotton and characterized. Expression analysis revealed that GhWRKY25 can be induced or deduced by the treatments of abiotic stresses and multiple defense-related signaling molecules. Overexpression of GhWRKY25 in Nicotiana benthamiana reduced plant tolerance to drought stress but enhanced tolerance to salt stress. Moreover, more MDA and ROS accumulated in transgenic plants after drought treatment with lower activities of SOD, POD, and CAT. Our study further demonstrated that GhWRKY25 overexpression in plants enhanced sensitivity to the fungal pathogen Botrytis cinerea by reducing the expression of SA or ET signaling related genes and inducing the expression of genes involved in the JA signaling pathway. These results indicated that GhWRKY25 plays negative or positive roles in response to abiotic stresses, and the reduced pathogen resistance may be related to the crosstalk of the SA and JA/ET signaling pathways.

Molecular cloning and functional characterisation of the tomato E3 ubiquitin ligase SlBAH1 gene.

Emerging evidence suggests that E3 ligases play critical roles in diverse biological processes, including pathogen resistance in plants. In the present study, an ubiquitin ligase gene (SlBAH1) was cloned from a tomato plant, and the functions of the gene were studied. The SlBAH1 gene contained 1002 nucleotides and encodes a protein with 333 amino acids. The SlBAH1 protein contains a SPX domain and a RING domain. SlBAH1 displayed E3 ubiquitin ligase activity in vitro. SlBAH1 was shown to localise in the nucleus, cytoplasm and plasma membrane by a subcellular localisation assay. The expression of SlBAH1 was induced by various hormones and Botrytis cinerea Pers. treatment. SlBAH1-silencing in plants obtained by virus-induced gene silencing (VIGS) technology enhanced resistance to B. cinerea, and the expression of pathogenesis-related (PR) genes, including PR1, PR2, PR4, PR5, and PR7, was significantly increased. These results indicate that the SlBAH1-dependent activation of defence-related genes played a key role in the enhanced fungal resistance observed in the SlBAH1-silenced plants and may be related to the SA-dependent and JA-dependent signalling pathways.

[Genetic analysis of RNA-mediated virus resistance in transgenic tobacco].

RNA-mediated virus resistance is an effective way to obtain virus resistant plants and is regarded as a potential strategy with application value in plant resistant virus breeding because of its advantage of high biosafety and long duration. Whether the resistance can inherit stably is a critical factor affecting its application in agriculture. In this paper, several T0 progeny transgenic plants with different resistant levels and with different transgene copy numbers were selected for further study. The results showed that the inheritance of the transgene in T0 susceptible transgenic plants containing 1 -2 transgene copies followed Mendelian segregation pattern of single gene inheritance in their progenies. Resistant transgenic plants, which contained 4 -6 transgene copies, followed 15:1 and 63:1 segregation patterns in the T1 progeny. Transgene recombination and re-organization in the plant genome was observed, which resulted in unstable inheritance of the transgene in the progenies. Homozygous resistant transgenic plants were obtained after 3 to 4 generations in the progeny. Analysis of the transgene integration pattern indicated that most of the highly resistant plants contained inverted repeat (IR) sequences of the transgene.

Effect of cDNA fragments in different length derived from potato virus Y coat protein gene on the induction of RNA-mediated virus resistance.

We have reported that cDNA derived from entire coat protein (CP) gene of potato virus Y (PVY) could induce resistance to PVY infection in transgenic tobacco plants, and the resistance was further demonstrated to be RNA-mediated rather than coat protein-mediated. In this study, we cloned cDNA fragments of 202 bp, 417 bp, and 603 bp in length derived from the 3' end of the PVY CP gene, and the cDNA fragments were introduced into tobacco (var. NC89) plants via Agrobacterium-mediated transformation system. The results of resistance assay showed that the CP cDNA fragments of 417 bp and 603 bp could confer resistance of the transgenic plants to PVY infection, but the fragment of 202 bp in length could not. Molecular analysis revealed that the resistance was RNA-mediated, which is believed to be a result of post-transcriptional gene silencing. The results indicate that the length of cDNA fragments needed for resistance induction was located somewhere between 202 bp and 417 bp from the 3' end of PVY CP gene.

Effects of the sequence characteristics of miRNAs on multi-viral resistance mediated by single amiRNAs in transgenic tobacco.

Artificial microRNA (amiRNA) has become the preferred viral defence that can be induced in plants. In this study, nine amiRNA target sites were selected that were based on the sequence characteristics of natural miRNAs in the cylindrical inclusion protein (CI), nuclear inclusion a protein (NIa), nuclear inclusion b protein (NIb), and coat protein (CP) genes of Potato virus Y (PVY(N)). These amiRNAs that exhibited high similarities to the sequences of PVY(N) and TEV-SD1 were considered. To study the effectiveness of gene silencing in amiRNA-mediated viral resistance, we constructed nine amiRNA plant expression vectors by replacing the functional sequences of miRNA319a precursors with our selected amiRNA sequences. These constructs were subsequently introduced to tobacco plants. A Northern blot assay verified that the nine amiRNA plant expression vectors could successfully express amiRNAs in plants. The analysis of viral resistance demonstrated that these transgenic tobacco plants could effectively inhibit PVY(N) and TEV-SD1 viral infections. The amiRNA that targeted the NIb and CP genes displayed a higher silencing efficiency than did the amiRNAs targeted CI and NIa genes. Northern blot analysis demonstrated that silencing was induced by the original amiRNAs and could be bilaterally extended by the siRNA pathway. That is, the amiRNA and the secondary siRNA mediated the degradation of viral RNA together. Genetic analysis demonstrated that the trait for viral resistance in transgenic plants can be consistently inherited via a single copy of the transgenic sequence. Considering the correlation between the sequence characteristics and the activity of amiRNA, we concluded that a few mismatched bases between the amiRNA and the target sequence could be allowed, particularly the mismatched bases in the 3' end of the amiRNA.

Production of marker-free and RSV-resistant transgenic rice using a twin T-DNA system and RNAi.

A twin T-DNA system is a convenient strategy for creating selectable marker-free transgenic plants. The standard transformation plasmid, pCAMBIA 1300, was modified into a binary vector consisting of two separate T-DNAs, one of which contained the hygromycin phosphotransferase (hpt) marker gene. Using this binary vector, we constructed two vectors that expressed inverted-repeat (IR) structures targeting the rice stripe virus (RSV) coat protein (CP) gene and the special-disease protein (SP) gene. Transgenic rice lines were obtained via Agrobacterium-mediated transformation. Seven independent clones harbouring both the hpt marker gene and the target genes (RSV CP or SP) were obtained in the primary transformants of pDTRSVCP and pDTRSVSP, respectively. The segregation frequencies of the target gene and the marker gene in the T1 plants were 8.72 percent for pDTRSVCP and 12.33 percent for pDTRSVSP. Two of the pDTRSVCP lines and three pDTRSVSP lines harbouring the homozygous target gene, but not the hpt gene, were strongly resistant to RSV. A molecular analysis of the resistant transgenic plants confirmed the stable integration and expression of the target genes. The resistant transgenic plants displayed lower levels of the transgene transcripts and specific small interfering RNAs, suggesting that RNAi induced the viral resistance.

Bacterially expressed double-stranded RNAs against hot-spot sequences of tobacco mosaic virus or potato virus Y genome have different ability to protect tobacco from viral infection.

Posttranscriptional gene silencing, also known as RNA interference, involves degradation of homologous mRNA sequences in organisms. In plants, posttranscriptional gene silencing is part of a defense mechanism against virus infection, and double-stranded RNA is the pivotal factor that induces gene silencing. In this paper, we got seven hairpin RNAs (hpRNAs) constructs against different hot-spot sequences of Tobacco mosaic virus (TMV) or Potato virus Y (PVY) genome. After expression in Escherichia coli HT115, we extracted the seven hpRNAs for the test in tobacco against TMV or PVY infection. The data suggest that different hpRNAs against different hot-spot sequences of TMV or PVY genome had different ability to protect tobacco plants from viral infection. The resistance to TMV conferred by the hpRNA against the TMV movement protein was stronger than other TMV hpRNAs; the resistance to PVY conferred by the hpRNA against the PVY nuclear inclusion b was better than that induced by any other PVY hpRNAs. Northern blotting of siRNA showed that the resistance was indeed an RNA-mediated virus resistance.