Yuwen Yang

Island Cotton Gbve1 Gene Encoding A Receptor-Like Protein Confers Resistance to Both Defoliating and Non-Defoliating Isolates of Verticillium dahliae

Verticillium wilt caused by soilborne fungus Verticillium dahliae could significantly reduce cotton yield. Here, we cloned a tomato Ve homologous gene, Gbve1, from an island cotton cultivar that is resistant to Verticillium wilt. We found that the Gbve1 gene was induced by V. dahliae and by phytohormones salicylic acid, jasmonic acid, and ethylene, but not by abscisic acid. The induction of Gbve1 in resistant cotton was quicker and stronger than in Verticillium-susceptible upland cotton following V. dahliae inoculation. Gbve1 promoter-driving GUS activity was found exclusively in the vascular bundles of roots and stems of transgenic Arabidopsis. Virus-induced silencing of endogenous genes in resistant cotton via targeting a fragment of the Gbve1 gene compromised cotton resistance to V. dahliae. Furthermore, we transformed the Gbve1 gene into Arabidopsis and upland cotton through Agrobacterium-mediated transformation. Overexpression of the Gbve1 gene endowed transgenic Arabidopsis and upland cotton with resistance to high aggressive defoliating and non-defoliating isolates of V. dahliae. And HR-mimic cell death was observed in the transgenic Arabidopsis. Our results demonstrate that the Gbve1 gene is responsible for resistance to V. dahliae in island cotton and can be used for breeding cotton varieties that are resistant to Verticillium wilt.

Comparative Transcriptome Profiling of a Resistant vs. Susceptible Tomato (Solanum lycopersicum) Cultivar in Response to Infection by Tomato Yellow Leaf Curl Virus

Tomato yellow leaf curl virus (TYLCV) threatens tomato production worldwide by causing leaf yellowing, leaf curling, plant stunting and flower abscission. The current understanding of the host plant defense response to this virus is very limited. Using whole transcriptome sequencing, we analyzed the differential gene expression in response to TYLCV infection in the TYLCV-resistant tomato breeding line CLN2777A (R) and TYLCV-susceptible tomato breeding line TMXA48-4-0 (S). The mixed inoculated samples from 3, 5 and 7 day post inoculation (dpi) were compared to non-inoculated samples at 0 dpi. Of the total of 34831 mapped transcripts, 209 and 809 genes were differentially expressed in the R and S tomato line, respectively. The proportion of up-regulated differentially expressed genes (DEGs) in the R tomato line (58.37%) was higher than that in the S line (9.17%). Gene ontology (GO) analyses revealed that similar GO terms existed in both DEGs of R and S lines; however, some sets of defense related genes and their expression levels were not similar between the two tomato lines. Genes encoding for WRKY transcriptional factors, R genes, protein kinases and receptor (-like) kinases which were identified as down-regulated DEGs in the S line were up-regulated or not differentially expressed in the R line. The up-regulated DEGs in the R tomato line revealed the defense response of tomato to TYLCV infection was characterized by the induction and regulation of a series of genes involved in cell wall reorganization, transcriptional regulation, defense response, ubiquitination, metabolite synthesis and so on. The present study provides insights into various reactions underlining the successful establishment of resistance to TYLCV in the R tomato line, and helps in the identification of important defense-related genes in tomato for TYLCV disease management.

Genome-wide analysis of tomato long non-coding RNAs and identification as endogenous target mimic for microRNA in response to TYLCV infection

Recently, a large number of long noncoding RNAs (lncRNAs) have emerged as important regulators of many biological processes in animals and plants. However, how lncRNAs function during plant DNA virus infection is largely unknown. We performed strand-specific paired-end RNA sequencing of tomato samples infected with Tomato yellow leaf curl virus (TYLCV) with three biological replicates. Overall, we predicted 1565 lncRNAs including long intergenic ncRNAs (lincRNAs) and natural antisense transcripts (lncNATs) and definitively identified lnRNAs that are involved in TYLCV infection by virus-induced gene silencing (VIGS). We also verified the functions of a set of lncRNAs that were differentially expressed between 0 and 7 days post inoculation (dpi). More importantly, we found that several lncRNAs acted as competing endogenous target mimics (eTMs) for tomato microRNAs involved in the TYLCV infection. These results provide new insight into lncRNAs involved in the response to TYLCV infection that are important components of the TYLCV network in tomatoes.

Genome-wide analysis of bHLH transcription factor and involvement in the infection by yellow leaf curl virus in tomato (Solanum lycopersicum)

BackgroundThe basic helix-loop-helix (bHLH) proteins are a superfamily of transcription factors that can bind to specific DNA target sites. They have been well characterized in model plants such as Arabidopsis and rice and have been shown to be important regulatory components in many different biological processes. However, no systemic analysis of the bHLH transcription factor family has yet been reported in tomatoes. Tomato yellow leaf curl virus (TYLCV) threatens tomato production worldwide by causing leaf yellowing, leaf curling, plant stunting and flower abscission.ResultsA total of 152 bHLH transcription factors were identified from the entire tomato genome. Phylogenetic analysis of bHLH domain sequences from Arabidopsis and tomato facilitated classification of these genes into 26 subfamilies. The evolutionary and possible functional relationships revealed during this analysis are supported by other criteria, including the chromosomal distribution of these genes, the conservation of motifs and exon/intron structural patterns, and the predicted DNA binding activities within subfamilies. Distribution mapping results showed bHLH genes were localized on the 12 tomato chromosomes. Among the 152 bHLH genes from the tomato genome, 96 bHLH genes were detected in the TYLCV-susceptible and resistant tomato breeding line before (0 dpi) and after TYLCV (357 dpi) infection. As anticipated, gene ontology (GO) analysis indicated that most bHLH genes are related to the regulation of macromolecule metabolic processes and gene expression. Only four bHLH genes were differentially expressed between 0 and 357 dpi. Virus-induced gene silencing (VIGS) of one bHLH genes SlybHLH131 in resistant lines can lead to the cell death.ConclusionIn the present study, 152 bHLH transcription factor genes were identified. One of which bHLH genes, SlybHLH131, was found to be involved in the TYLCV infection through qRT-PCR expression analysis and VIGS validation. The isolation and identification of these bHLH transcription factors facilitated clarification of the molecular genetic basis for the genetic improvement of tomatoes and the development of functional gene resources for transgenic research. In addition, these findings may aid in uncovering an unexplored mechanism during the TYLCV infection in tomatoes.

A Cotton Gbvdr5 Gene Encoding a Leucine-Rich-Repeat Receptor-Like Protein Confers Resistance to Verticillium dahliae in Transgenic Arabidopsis and Upland Cotton

Leucine-rich-repeat receptor-like proteins (eLRR-RLPs) play significant roles in plant defense against pathogens and in plant development. Several eLRR-RLP genes such as Ve1, Gbve, and Gbve1 have been reported to confer resistance to Verticillium dahliae. Gbvdr5, a newly discovered RLP gene from V. dahliae-resistant island cotton cultivar H7124, has a short tail as Ve1. There is a cytosine deletion in Gbvdr5 homologous genes at nucleotide position 2765, which is downstream from the initiation codon in all susceptible upland cotton cultivars analyzed. This deletion was found to cause premature termination of the protein, creating a 937 aa product, but the Gbvdr5 protein had the full 1,077 aa. Transient expression analyses indicated that Gbvdr5 is localized on the plasma membrane. Quantitative polymerase chain reaction analysis revealed that the Gbvdr5 gene was activated by SA, MeJA, ABA, and ETH, and it was induced by V. dahliae isolates V991 and DF-CQ-2 in H7124, whereas was unchanged or repressed in susceptible upland cotton Simian 3. Gbvdr5-promoter-driven GUS activity was found mostly in the root tips and stem growing points of transgenic Arabidopsis. Silencing of Gbvdr5 in Verticillium-wilt-resistant cotton H7124 compromised cotton resistance to V. dahliae isolates V991 and BP2. The resistance was verified by transforming the Gbvdr5 gene into Arabidopsis and upland cotton through Agrobacterium-mediated transformation. Overexpression of the Gbvdr5 gene endowed transgenic Arabidopsis with resistance to defoliating isolate V991 and non-defoliating isolate BP2, but it had no effect on either DF-CQ-2 or JR2 of V. dahliae. The transformed cotton also had confirmed resistance to V991 and BP2. More callose deposition, more expression of the defense-related genes PR1 and PR5, and HR-mimic cell death were observed in the transgenic Arabidopsis when inoculated with V. dahliae. This demonstrated that Verticillium–plant interactions may involve some specific ways of recognizing V. dahliae and Gbvdr5 may be a suitable candidate gene for breeding Verticillium-wilt-resistant cotton lines.

Cotton Leaf Curl Multan Virus-Derived Viral Small RNAs Can Target Cotton Genes to Promote Viral Infection

RNA silencing is a conserved mechanism in plants that targets viruses. Viral small RNAs (vsiRNAs) can be generated from viral double-stranded RNA replicative intermediates within the infected host, or from host RNA-dependent RNA polymerases activity on viral templates. The abundance and profile of vsiRNAs in viral infections have been reported previously. However, the involvement of vsiRNAs during infection of the Geminiviridae family member cotton leaf curl virus (CLCuD), which causes significant economic losses in cotton growing regions, remains largely uncharacterized. Cotton leaf curl Multan virus (CLCuMuV) associated with a betasatellite called Cotton leaf curl Multan betasatellite (CLCuMuB) is a major constraint to cotton production in South Asia and is now established in Southern China. In this study, we obtained the profiles of vsiRNAs from CLCuMV and CLCuMB in infected upland cotton (Gossypium hirsutum) plants by deep sequencing. Our data showed that vsiRNA that were derived almost equally from sense and antisense CLCuD DNA strands accumulated preferentially as 21- and 22-nucleotide (nt) small RNA population and had a cytosine bias at the 5′-terminus. Polarity distribution revealed that vsiRNAs were almost continuously present along the CLCuD genome and hotspots of sense and antisense strands were mainly distributed in the Rep proteins region of CLCuMuV and in the C1 protein of CLCuMuB. In addition, hundreds of host transcripts targeted by vsiRNAs were predicted, many of which encode transcription factors associated with biotic and abiotic stresses. Quantitative real-time polymerase chain reaction analysis of selected potential vsiRNA targets showed that some targets were significantly down-regulated in CLCuD-infected cotton plants. We also verified the potential function of vsiRNA targets that may be involved in CLCuD infection by virus-induced gene silencing (VIGS) and 5′-rapid amplification of cDNA end (5′-RACE). Here, we provide the first report on vsiRNAs responses to CLCuD infection in cotton.

A CC-NBS-LRR type gene GHNTR1 confers resistance to southern root-knot nematode in Nicotiana.benthamiana and Nicotiana.tabacum

Root-knot nematodes are obligate parasites that invade the roots of agricultural plants and induce the formation of specialized feeding structures, especially races 3 and 4 of the southern root-knot nematode. However, not much is known about the defense mechanisms of plants against the invasion of M. incognita race 1. In this study, we characterized and performed functional analysis of the CC-NBS-LRR domain gene, GHNTR1. Using the GHNTR1 promoter to drive the GUS marker gene, we found that GUS expression was high in the roots and shoots of seedlings, four leaves stages, and mature stages. When GHNTR1 was transiently expressed in Nicotiana. benthamiana and Nicotiana. tabacum, necrosis was observed in the leaves and detectable amounts of H2O2 had accumulated when compared to the control plants. Stable transformation of N. benthamiana with the GHNTR1 gene using Agrobacterium induced the expression of defense marker genes PR1, PR2, LOX, and ERF1. In addition, the transgenic N. benthamiana and N. tabacum plants exhibited higher resistance to M. incognita infection. When transgenic N. tabacum were challenged with M. incognita, callose deposition and peroxide accumulation were observed in the roots by aniline-blue and DAB staining, respectively. Sequencing and bioinformatic analysis of small RNA revealed 48 miRNAs were up-regulated and 32 miRNAs were down-regulated. Further, 345 genes were predicted as targets of miRNAs and these included genes that are involved in cell death, death, and stress response. Thus, GHNTR1 is the first gene isolated from cotton that confers resistance against M. incognita and it induced a series of hypersensitive responses in transgenic tobacco. These findings improve our understanding of the molecular mechanisms of the response of upland cotton to M. incognita infection.

A P4-ATPase Gene GbPATP of Cotton Confers Chilling Tolerance in Plants

Members of the P4 subfamily of P-type ATPases are implicated in generating lipid asymmetry between the two lipid leaflets of the plasma membrane in Arabidopsis and are important for resistance to low temperatures, but the function of P4-ATPases in cotton remains unclear. In this study, we found using quantitative reverse transcription-PCR analysis that the expression of the P4-ATPase gene GbPATP in cotton was induced at low temperatures. In addition, GbPATP-silenced cotton plants were more sensitive to low temperatures and exhibited greater malondialdehyde (MDA) content and lower catalase (CAT) activity than the control plants. GbPATP transgenic tobacco plants showed better chilling tolerance, had a lower MDA content and had higher CAT activity than wild-type plants under low-temperature treatment. The green fluorescent protein (GFP)-GbPATP fusion protein was found to be localized to the cell plasma membrane. Collectively, the results suggest that GbPATP functions as a P4-ATPase and plays an important role in improving chilling tolerance in plant.

Two Lysin-Motif Receptor Kinases, Gh-LYK1 and Gh-LYK2, Contribute to Resistance against Verticillium wilt in Upland Cotton

Lysin-motif (LysM) receptor kinases (LYKs) play essential roles in recognition of chitin and activation of defense responses against pathogenic fungi in the model plants Arabidopsis and rice. The function of LYKs in non-model plants, however, remains elusive. In the present work, we found that the transcription of two LYK-encoding genes from cotton, Gh-LYK1 and Gh-LYK2, was induced after Verticillium dahliae infection. Virus-induced gene silencing (VIGS) of Gh-LYK1 and Gh-LYK2 in cotton plants compromises resistance to V. dahliae. As putative pattern recognition receptors (PRRs), both Gh-LYK1 and Gh-LYK2 are membrane-localized, and all three LysM domains of Gh-LYK1 and Gh-LYK2 are required for their chitin-binding ability. However, since Gh-LYK2, but not Gh-LYK1, is a pseudo-kinase and, on the other hand, the ectodomain (ED) of Gh-LYK2 can induce reactive oxygen species (ROS) burst in planta, Gh-LYK2 and Gh-LYK1 may contribute differently to cotton defense. Taken together, our results establish that both Gh-LYK1 and Gh-LYK12 are required for defense against V. dahliae in cotton, possibly through different mechanisms.

Microscopic observations of strawberry plant colonization by a GFP-labelled strain of Fusarium oxysporum f. sp. fragariae

Abstract Fusarium oxysporum f. sp. fragariae, the causal agent of Fusarium wilt in strawberry (Fragaria ananassa Duch.), is a serious threat to commercial production. To understand the interaction between strawberry and Fusarium oxysporum f. sp. fragariae, we transformed the pathogen with a green fluorescent protein as a labelled gene and Hyg as a selectable marker gene using the Agrobacterium tumefaciens-mediated transformation method. The transformants obtained were stable with strong and uniform fluorescence. A green fluorescent protein-tagged strain with similar phenotype and pathogenicity as the wild-type strain was used to study the infection process on strawberry. The observations indicated that the spores attached to the root surface and infected the roots from germ tubes, while the hyphae produced suction cup-like structures from the hyphal tips to infect the strawberry plants. Once inside, hyphal growth was observed mainly in the epidermal and cortical tissues with only a few hyphae detected in the vascular tissues after colonization.