A novel transcription factor FnMYB4 regulates pigments metabolism of yellow leaf mutants in Fragaria nilgerrensis

The strawberry species Fragaria nilgerrensis Schlechtendal ex J.Gay,renowned for its distinctive white,fragrant peach-like fruits and strong disease resistance,is an exceptional research material.In a previous study,an ethyl methane sulfonate(EMS)mutant library was established for this species,resulting in various yellow leaf mutants.Leaf yellowing materials are not only the ideal materials for basic studies on photosynthesis mechanism,chloroplast development,and molecular regulation of various pigments,but also have important utilization value in ornamental plants breeding.The present study focused on four distinct yellow leaf mutants:mottled yellow leaf(MO),yellow green leaf(YG),light green leaf(LG),and buddha light leaf(BU).The results revealed that the flavonoid content and carotenoid-to-chlorophyll ratio exhibited a significant increase among these mutants,while experiencing a significant decrease in chlorophyll and carotenoid contents compared to the wild type(WT).To clarify the regulatory mechanisms and network relationships underlying these mutants,the RNA-seq and weighted gene coexpression network(WGCNA)analyses were employed.The results showed flavonoid metabolism pathway was enriched both in MO and YG mutants,while the chlorophyll biosynthesis pathway and carotenoid degradation pathway were only enriched in MO and YG mutants,respectively.Subsequently,key structural genes and transcription factors were identified on metabolic pathways of three pigments through correlation analyses and quantitative experiments.Furthermore,a R2R3-MYB transcription factor,FnMYB4,was confirmed to be positively correlated with flavonoid synthesis through transient overexpression,virus-induced gene silencing(VIGS),and RNA interference(RNAi),accompanying by reoccurrence and attenuation of mutant phenotype.Finally,dual-luciferase(LUC)and yeast one-hybrid assays confirmed the binding of FnMYB4 to the FnFLS and FnF3H promoters,indicating that FnMYB4 positively regulates flavonoid synthesis.In addition,correlation analyses suggested that FnMYB4 also might be involved in chlorophyll and carotenoid metabolisms.These findings demonstrated the pivotal regulatory role of FnMYB4 in strawberry leaf coloration.

Detection and Identification of Phytoplasma of Cleome rutidosperma in Areca Palm Yellow Leaf Disease Field

[Objectives]The paper was to detect and identify the phytoplasma of Cleome rutidosperma in areca palm yellow leaf disease(YLD)field in Wenchang City,Hainan Province,China.[Methods]The nested PCR technique was employed to amplify the phytoplasma 16S rDNA of C.rutidosperma samples,followed by sequence analysis.Concurrently,this study examined C.rutidosperma in YLD field,collecting symptomatic leaves for phytoplasma detection.[Results]The 16S rDNA sequence of the C.rutidosperma witches'-broom phytoplasma was found to be identical to that of the HNWC5 strain associated with areca palm yellows phytoplasma,leading to the identification of this phytoplasma as belonging to the 16SrII-A subgroup.Field investigations revealed a higher incidence of C.rutidosperma in areca palm fields,with symptoms of leaf yellows observed in six of these fields.Quantitative PCR(qPCR)analysis confirmed the presence of phytoplasma infection in these instances.[Conclusions]Through the analysis of geographical distribution,sequence alignment,and field occurrence data,a significant correlation has been identified between witches'broom disease and YLD.It is proposed that the former may act as an intermediate host for the areca palm yellows phytoplasma.

Diverse Regulations of Viral and Host Genes in Tomato Germplasms Responding to Tomato Yellow Leaf Curl Virus Inoculation

Tomato yellow leaf curl virus(TYLCV)is the dominating pathogen of tomato yellow leaf curl disease that caused severe loss to tomato production in China.In this study,we found that a TYLCV-resistant tomato line drastically reduced the accumulation of viral complementary-sense strand mRNAs but just moderately inhibited that of viral DNA and virion-sense strand mRNAs.However,two other resistant lines did not have such virus inhibition pattern.Analysis of differential expressed genes showed that the potential host defense-relevant processes varied in different resistant tomatoes,as compared to the susceptible line,suggesting a diversity of tomato TYLCV-resistance mechanisms.

Monoclonal Antibodies Against the Whitefly-Transmitted Tomato Yellow Leaf Curl Virus and Their Application in Virus Detection

Tomato yellow leaf curl virus（TYLCV）is a species of the family Geminiviridae,causing serious yield losses in tomato production.The coat protein（CP）gene of TYLCV isolate SH2 was expressed in Escherichia coli BL21（DE3）using pET-32a as the expression vector.The recombinant protein was purified through Ni＋-NTA affinity column and used to immunize BALB/c mice.Three hybridoma cell lines（2B2,2E3 and 3E10）secreting monoclonal antibodies（MAbs）against TYLCV CP were obtained by fusing mouse myeloma cells（Sp 2/0）with spleen cells from the immunized BALB/c mouse.The titers of ascitic fluids of three MAbs ranged from 10-6 to 10-7 in indirect-ELISA.Isotypes and subclasses of all the MAbs belonged to IgG1,κ light chain.Triple antibody sandwich enzyme-linked immunosorbent assay（TAS-ELISA）showed that the MAb 3E10 could react with five begomoviruses infecting tomato,while the other two（2B2 and 2E3）mainly reacted with TYLCV.TAS-ELISA was set up using the MAb 3E10,and the established method could successfully detect virus in plant sap at 1：2 560（w/v,g mL-1）.Detection of field samples showed that begomoviruses were common in tomato crops in Zhejiang Province,China.

A New Disease of Cherry Plum Tree with Yellow Leaf Symptoms Associated with a Novel Phytoplasma in the Aster Yellows Group

A novel phytoplasma was detected in a cherry plum（Prunus cerasifera Ehrh） tree that mainly showed yellow leaf symptom. The tree was growing in an orchard located in Yangling District, Shaanxi Province, China. The leaves started as chlorotic and yellowing along leaf minor veins and leaf tips. Chlorosis rapidly developed to inter-veinal areas with the whole leaf becoming pale yellow in about 1-4 wk. Large numbers of phytoplasma-like bodies（PLBs） were seen under transmission electron microscopy. The majority of the PLBs was spherical or elliptical vesicles, with diameters in range of 0.1-0.6 μm, and distributed in the phloem cells of the infected tissues. A 1 246-bp 16 S ribosomal RNA（rRNA） gene fragment was amplified from DNA samples extracted from the yellow leaf tissues using two phytoplasma universal primer pairs R16mF2/R16mR1 and R16F2n/R16R2. Phylogenetic analysis using the 16 S rRNA gene sequence suggested that the phytoplasma associated with the yellow leaf symptoms belongs to a novel subclade in the aster yellows（AY） group（16SrI group）. Virtual and actual restriction fragment length polymorphism（RFLP） analysis of the 16 S rRNA gene fragment revealed that the phytoplasma was distinguishable from all existing 19 subgroups in the AY group（16SrI） by four restriction sites, Hinf I, Mse I, Sau3 A I and Taq I. The similarity coefficients of comparing the RFLP pattern of the 16 S rRNA gene fragment of this phytoplasma to each of the 19 reported subgroups ranged from 0.73 to 0.87, which indicates the phytoplasma associated with the cherry plum yellow leaf（CPYL） symptoms is probably a distinct and novel subgroup lineage in the AY group（16SrI）. In addition, the novel phytoplasma was experimentally transmitted to periwinkle（Catharanthus roseus） plants from the tree with CPYL symptoms and then back to a healthy 1-yr-old cherry plum tree via dodder（Cuscuta odorata） connections.

Development of high yield and tomato yellow leaf curl virus(TYLCV)resistance using conventional and molecular approaches:A review

Tomato(Solanum lycopersicum L.)belonging to the family Solanaceae is the second most consumed and cultivated vegetable globally.Since the ancient time of its domestication,thousands of cultivated tomato varieties have been developed targeting an array of aspects.Among which breeding for yield and yield-related traits are mostly focused.Cultivated tomato is extremely genetically poor and hence it is a victim for several biotic and abiotic stresses.Among the biotic stresses,the impact of viral diseases is critical all over tomato cultivating areas.Improvement of tomato still largely rely on conventional methods worldwide while molecular approaches,particularly Marker Assisted Selection(MAS)has become popular across the globe as a fast,low cost and precise tool which is essential in present day plant breeding.In this review paper,breeding tomato for high yield and viral disease resistance,particularly to tomato yellow leaf curl virus disease(TYLCVD)using conventional and molecular approaches will be discussed.Lining up of this set of information will be useful to those who are interested in tomato variety development with high yielding and TYLCVD resistance.

Mapping and Candidate Gene Screening of Tomato Yellow Leaf Curl Virus Resistant Gene ty-5

To identify the inheritance pattern and perform fne mapping of ty-5 gene, P1, P2, F1, BC1 and F2 generations were obtained through a cross between CLN32120a-23 （containing ty-5 gene, P1） and S. lycopersicum Moneymaker （fully susceptible, P2）. The results showed that resistance of ty-5 gene was determined by a recessive effect. Meanwhile, it was presumed that another resistance gene might be involved in mediating the resistance to tomato yellow leaf curl virus （TYLCV）. In this study, fne mapping was used to map TYLCV resistance locus to an interval between NAC1 and TES2461 on the short arm of chromosome 4 with genetic distances of 0.5 and 0.8 cM, respectively. qRT-PCR results showed that four candidate genes, SlNAC1; LOC104229164; LOC101260925 and LOC101261508 having resistance-related expression patterns, were the likely target genes of ty-5. In addition, it was found that the codominant marker TES2461 could be used in marker-assisted selection （MAS） breeding. The fndings of this research provided the basis for future cloning of ty-5 gene as well as MAS breeding and plant resistance mechanism studies.

Characterization and function of Tomato yellow leaf curl virus-derived small RNAs generated in tolerant and susceptible tomato varieties

supported by the Specialized Research Fund for the Doctoral Program of Higher Education of China （20134320120013）;the Natural Science Foundation of Hunan Province, China （14JJ3095）

Science Letters:Expression of a begomoviral DNAβ gene in transgenic Nicotiana plants induced abnormal cell division

An increasing number of monopartite begomoviruses are being identified that a satellite molecule (DNAβ) is required to induce typical symptoms in host plants. DNAβ encodes a single gene (termed βC1) encoded in the complementary-sense. We have produced transgenic Nicotiana benthamiana and N. tabacum plants expressing theβC1 gene of a DNAβ associated with Tomato yellow leaf curl China virus (TYLCCNV), under the control of the Cauliflower mosaic virus 35S promoter. Transgenic plants expressing βC1 showed severe developmental abnormalities in both species. Microscopic analysis of sections of both transgenic and non-transgenic N. tabacum leaves showed abnormal outgrowths of transgenic N. tabacum to be due to disorganized cell division (hyperplasia) of spongy and palisade parenchyma. Immuno-gold labeling of sections with a polyclonal antibody against the βC1 protein showed that the βC1 protein accumulated in the nuclei of cells. The possible biological function of the βC1 protein was discussed.

Biomimetic Material Simulating Solar Spectrum Reflection Characteristics of Yellow Leaf

To counter the threat of hyperspectral detection, it is necessary to develop biomimetic materials to simulate the solar spectral re- flection characteristics of plant leaf accurately. Two kinds ofmembranaceous yellow biomimetic materials were prepared by dispersing the particles of chrome titanium yellow and iron oxide yellow as fillers in polyvinyl alcohol films respectively. Reflectance and transmittance of the biomimetic materials were measured, and absorption and scattering coefficients of the biomimetic materials were inverted with a four-flux model. Results indicate that the biomimetic material adopting chrome titanium yellow particles can simulate the solar spectrum reflection characteristics of yellow leaf because of the similar absorption and scattering characteristics. The biomimetic material adopting iron oxide yellow particles cannot simulate the spectrum reflection characteristics of yellow leaf near the wavelength of 900 nm due to the characteristic absorption of the iron oxide. When the volume fraction of the chrome titanium yellow particles is lower than 2.12%, the absorption and scattering coefficients both increase approximately linearly with the volume fraction, indicating that the particles can scatter radiation independently. Therefore, the reflectance of the biomimetic material can be regulated through linearly changing of the volume fraction of the chrome titanium yellow particles.

Tomato yellow leaf curl virus infection of tomato does not affect the performance of the Q and ZHJ2 biotypes of the viral vector Bemisia tabaci

To better understand the etiology of begomovirus epidemics in regions under invasion we need to know how indigenous and invasive whitefly vectors respond to virus infection. We investigated both direct and indirect effects of infection with Tomato yellow leaf curl virus （TYLCV） on the performance of the invasive Q biotype and the indigenous Asian ZHJ2 biotype of whitefly Bemisia tabaci. The Q biotype performed better than the ZHJ2 biotype on either uninfected or virus-infected tomato plants. However, virus-infection of host plants did not, or only marginally affected, the performance of either biotype of whiteflies in terms of fecundity, longevity, survival, development and population increase. Likewise, association of the vectors with TYLCV did not affect fecundity and longevity of the Q or ZHJ2 biotypes on cotton, a non-host of TYLCV. These results indicate that the alien Q biotype whitefly, but not the indigenous ZHJ2 biotype, is likely to become the major vector of TYLCV in the field and facilitate virus epidemics.

The novel C5 protein from tomato yellow leaf curl virus is a virulence factor and suppressor of gene silencing

Tomato yellow leaf curl virus(TYLCV)is known to encode 6 canonical viral proteins.Our recent study revealed that TYLCV also encodes some additional small proteins with potential virulence functions.The fifth ORF of TYLCV in the complementary sense,which we name C5,is evolutionarily conserved,but little is known about its expression and function during viral infection.Here,we confirmed the expression of the TYLCV C5 by analyzing the promoter activity of its upstream sequences and by detecting the C5 protein in infected cells by using a specific custom-made antibody.Ectopic expression of C5 using a potato virus X(PVX)vector resulted in severe mosaic symptoms and higher virus accumulation levels followed by a burst of reactive oxygen species(ROS)in Nicotiana benthamiana plants.C5 was able to effectively suppress local and systemic post-transcriptional gene silencing(PTGS)induced by single-stranded GFP but not double-stranded GFP,and reversed the transcriptional gene silencing(TGS)of GFP.Furthermore,the mutation of C5 in TYLCV inhibited viral replication and the development of disease symptoms in infected plants.Transgenic overexpression of C5 could complement the virulence of a TYLCV infectious clone encoding a dysfunctional C5.Collectively,this study reveals that TYLCV C5 is a pathogenicity determinant and RNA silencing suppressor,hence expanding our knowledge of the functional repertoire of the TYLCV proteome.

Screening of Growth-Promoting Strains for Areca Palm

[Objectives]The paper was to identify growth-promoting strains within the culturable bacterial flora of areca palm.[Methods]Culturable bacteria were isolated and identified from areca palm using samples obtained from both healthy and yellowing disease-affected plants within the same orchard.Strains that exhibited significant differences between healthy and affected samples,or that were unique to the healthy samples,were subsequently screened for their growth-promoting effects.[Results]Three bacterial strains demonstrated robust and consistent capacity for auxin production,specifically Paenibacillus,Pseudomonas aeruginosa,and Bacillus amyloliquefaciens,each yielding approximately 50μg of IAA per mL of bacterial solution.The strain Alcaligenes faecalis exhibited the highest efficacy in siderophore production,achieving 21.15%of active units.Additionally,A.faecalis,Bacillus velezensis,and P.aeruginosa were noted for their potassium-solubilizing capabilities,as evidenced by the presence of distinct potassium-solubilizing zones.[Conclusions]The evaluation of the aforementioned growth-promoting strains may offer valuable insights for the development of growth-promoting strains specifically for areca palm.

Molecular variability and evolution of a natural population of tomato yellow leaf curl virus in Shanghai,China

Tomato yellow leaf curl virus （TYLCV）, belonging to the genus Begomovirus of the family Geminiviridae, is emerging as the most destructive pathogen of tomato plants. Since the first report of TYLCV in Shanghai, China in 2006, TYLCV has spread rapidly to 13 provinces or autonomous regions of China. In this study, the molecular varia- bility and evolution of TYLCV were monitored in Shanghai from its first upsurge in 2006 until 2010. Full-length genomic sequences of 26 isolates were obtained by rolling circle amplification. Sequence analysis showed that the intergenic region was the most variable, with a mean mutation rate of 4.81×10-3 nucleotide substitutions per site per year. Ge- netic differentiation was found within isolates obtained from 2006, 2009, and 2010, though a linear increase in genetic diversity over time was not evident. Whilst significant parts of TYLCV genes were under negative selection, the C4 gene embedded entirely within the C1 gene had a tendency to undergo positive selection. Our results indicate that a mechanism of independent evolution of overlapping regions could apply to the natural population of TYLCV in Shanghai, China.

Efficiency for Gene Silencing Induction in Nicotiana Species by a Viral Satellite DNA Vector

Virus-induced gene silencing （VIGS） is a useful technique for rapid plant gene function analysis. We recently reported a new VIGS vector modified from Tomato yellow leaf curl China virus （TYLCCNV） DNAβ （DNAm β）. In this study we compared in detail DNAmβ-induced gene silencing in four Nicotiana species including N. benthamiana, N. glutinosa, N. tabacum and N. paniculata. We found that DNAmβ-induced gene silencing in the four species was distinct in developing dynamics, tissue specificity, efficiency, and constancy in the plant life span. It was most efficient in N. benthamiana, where development of VIGS was most rapid, without tissue specificity and nearly 100% efficient. DNAmβ-induced gene silencing in N. glutinosa was also efficient despite being slightly less than in N. benthamiana. It initially occurred in veins, later was scattered to mesophyll, finally led to complete silencing in whole leaves. In both species, VIGS constantly expressed until the plants died. However, DNAmβ-mediated VIGS in the other two Nicotiana species, N. tabacum and N. paniculata, was significantly less efficient. It was strictly limited within the veins of the silenced leaves, and constantly occurred only over 3-4 weeks. The upper leaves that emerged later stopped showing the silencing phenotype. DNAmβ-induced gene silencing in N. benthamiana and N. glutinosa was not significantly influenced by the growth stage when the plants were agro-inoculated, and was not sensitive to high growth temperature up to 32℃. Our results indicate that this system has great potential as a versatile VIGS system for routine functional analysis of genes in some Nicotiana species.