Complete genome sequence of a novel virga-like virus infecting Hevea brasiliensis.

Here, we report the complete genome sequence and organization of a novel virus detected in rubber trees (Hevea brasiliensis). Because the infected plants were asymptomatic, this virus was tentatively named "rubber tree latent virus 1" (RTLV1). The full genome of RTLV1 is 9,422 nt in length and contains three open reading frames with a 157-nt 5' untranslated region (UTR) and a 316-nt 3' UTR. The replicase shares the highest amino acid (aa) sequence identity (32.62%), with only 31% query coverage, with the replicase of Hubei virga-like virus 11. Phylogenetic analysis based on the aa sequence of ORF1 showed that RTLV1 clustered with unclassified members of the family Virgaviridae in a clade that was not closely related to any genus in this family.

A Phytochrome B-Independent Pathway Restricts Growth at High Levels of Jasmonate Defense.

The plant hormone jasmonate (JA) promotes resistance to biotic stress by stimulating the degradation of JASMONATE ZIM-DOMAIN (JAZ) proteins, which relieves repression on MYC transcription factors that execute defense programs. JA-triggered depletion of JAZ proteins in Arabidopsis (Arabidopsis thaliana) is also associated with reduced growth and seed production, but the mechanisms underlying these pleiotropic growth effects remain unclear. Here, we investigated this question using an Arabidopsis JAZ-deficient mutant (jazD; jaz1-jaz7, jaz9, jaz10, and jaz 13) that exhibits high levels of defense and strong growth inhibition. Genetic suppressor screens for mutations that uncouple growth-defense tradeoffs in the jazD mutant identified nine independent causal mutations in the red-light receptor phytochrome B (phyB). Unlike the ability of the phyB mutations to completely uncouple the mild growth-defense phenotypes in a jaz mutant (jazQ) defective in JAZ1, JAZ3, JAZ4, JAZ9, and JAZ10, phyB null alleles only weakly alleviated the growth and reproductive defects in the jazD mutant. phyB-independent growth restriction of the jazD mutant was tightly correlated with upregulation of the Trp biosynthetic pathway but not with changes in central carbon metabolism. Interestingly, jazD and jazD phyB plants were insensitive to a chemical inhibitor of Trp biosynthesis, which is a phenotype previously observed in plants expressing hyperactive MYC transcription factors that cannot bind JAZ repressors. These data provide evidence that the mechanisms underlying JA-mediated growth-defense balance depend on the level of defense, and they further establish an association between growth inhibition at high levels of defense and dysregulation of Trp biosynthesis.

Complete genome sequence of a novel capillovirus infecting Hevea brasiliensis in China.

Tapping panel dryness (TPD) is a complex disorder that causes partial or complete cessation of latex drainage upon tapping of rubber trees (Hevea brasiliensis). In this work, we determined the complete genome sequences of a novel virus identified in a rubber tree with TPD syndrome in China. The genome of the virus consists of 6811 nt and possesses two overlapping open reading frames (ORF1 and ORF2), encoding a polyprotein and a movement protein, respectively. The polyprotein shares 37% amino acid sequence identity with cherry virus A (CVA, ARQ83874.1) over 99% coverage. The genome architecture is similar to that of members of the genus Capillovirus (family Betaflexiviridae). Phylogenetic analysis of the replicase proteins showed that the virus clustered together with members of the genus Capillovirus. The new virus is tentatively called "rubber tree virus 1" (RTV1). RTV1 is the first virus reported to infect rubber trees. This work lays a foundation for research into finding the potential causal agent of TPD in Hevea brasiliensis.

Prevalence of Yellow Leaf Disease (YLD) and its Associated Areca Palm Velarivirus 1 (APV1) in Betel Palm (Areca catechu) Plantations in Hainan, China.

Yellow leaf disease (YLD) is an economically important disease affecting betel palm in several countries, the cause of which remains unclear despite associations with putative agents, including phytoplasmas. In this study, we screened the potential casual agents associated with YLD in Hainan, China using next-generation sequencing and revealed the association of areca palm velarivirus 1 (APV1) with the YLD-affected palm. The complete genome of the APV1-WNY isolate was determined to be 17,546 nucleotides in length, approximately 1.5 kb longer than the previously reported APV1_HN genome. Transmission electron microscopy showed that APV1 particles are flexuous and filamentous, a typical morphology of species in the Closteroviridae family. Comparison of symptomatic and symptomless tree populations showed a strong association between APV1 and YLD. APV1 was detected in Pseudococcus sp. mealybugs sampled from YLD-affected trees in many locations, suggesting that mealybugs are a potential transmission vector for APV1. Although further studies are needed to confirm a causal relationship, these results provide timely information for the prevention and management of YLD associated with APV1.

Identification of the targets of HbEIN3/EILs in genomic wide in Hevea brasiliensis.

EIN3/EILs are key regulators in ET signaling pathway. In this work, 4 members of EIN3/EILs of Hevea brasiliensis (HbEIN3/EILs) showed interaction with two F box proteins, HbEBF1 and HbEBF2. HbEIN3 located in nucleus and exhibited strong transcriptional activity. HbEIN3 was induced by ET treatment in C-serum, but not in B-serum of latex. HbEIN3/EILs bound to G-box cis-element. To globally search the potential targets of HbEIN3/EILs, genomic sequences of H. brasiliensis was re-annotated and an HCES (Hevea Cis-Elements Scanning) program was developed ( www.h-brasiliensis.com ). HCES scanning results showed that ET- and JA- responsive cis-elements distribute overlapping in gene promoters. 3146 genes containing G-box in promoters are potential targets of HbEIN3, including 41 genes involved in biosynthesis and drainage of latex, of which 7 rate-limiting genes of latex production were regulated by both ET and JA, suggesting that ET and JA signaling pathways coordinated the latex biosynthesis and drainage in H. brasiliensis. Abbreviations: ABRE: ABA responsive elements; bHLH: basic helix-loop-helix; COG: Orthologous Groups; DRE: dehydration response element; ERE: ethylene responsive element; ET: Ethylene; GO: Gene Ontology; HCES: Hevea Cis-Elements Scanning; JA: jasmonates; JRE: Jasmonate-responsive element; KEGG: Kyoto Encyclopedia of Genes and Genomes; NR: non-redundant database; PLACE: Plant Cis-acting Regulatory DNA Elements; qRT-PCR: quantitative real-time RT-PCR.

Function of Hevea brasiliensis NAC1 in dehydration-induced laticifer differentiation and latex biosynthesis.

MAIN CONCLUSIONS: HbNAC1 is a transcription factor in rubber plants whose expression is induced by dehydration, leading to latex biosynthesis. Laticifer is a special tissue in Hevea brasiliensis where natural rubber is biosynthesized and accumulated. In young stems of epicormic shoots, the differentiation of secondary laticifers can be induced by wounding, which can be prevented when the wounding site is wrapped. Using this system, differentially expressed genes were screened by suppression subtractive hybridization (SSH) and macroarray analyses. This led to the identification of several dehydration-related genes that could be involved in laticifer differentiation and/or latex biosynthesis, including a NAC transcription factor (termed as HbNAC1). Tissue sections confirmed that local tissue dehydration was a key signal for laticifer differentiation. HbNAC1 was localized at the nucleus and showed strong transcriptional activity in yeast, suggesting that HbNAC1 is a transcription factor. Furthermore, HbNAC1 was found to bind to the cis-element CACG in the promoter region of the gene encoding the small rubber particle protein (SRPP). Transgenic experiments also confirmed that HbNAC1 interacted with the SRPP promoter when co-expressed, and enhanced expression of the reporter gene ß-glucuronidase occurred in planta. In addition, overexpression of HbNAC1 in tobacco plants conferred drought tolerance. Together, the data suggest that HbNAC1 might be involved in dehydration-induced laticifer differentiation and latex biosynthesis.

Reprogramming of the Hevea brasiliensis Epigenome and Transcriptome in Response to Cold Stress.

Low temperature is a key factor limiting the rubber plantation extending to high latitude area. Previous work has shown that cold-induced DNA demethylation was coordinated with the expression of cold-responsive (COR) genes in Hevea brasiliensis. In this work, reduced representation bisulphite sequencing analysis of H. brasiliensis showed that cold treatment induced global genomic DNA demethylation and altered the sequence contexts of methylated cytosines, but the levels of mCG methylation in transposable elements were slightly enhanced by cold treatment. Integrated analysis of the DNA methylome and transcriptome revealed 400 genes whose expression correlated with altered DNA methylation. DNA demethylation in the upstream region of gene seems to correlate with higher gene expression, whereas demethylation in the gene body has less association. Our results suggest that cold treatment globally change the genomic DNA methylation status of the rubber tree, which might coordinate reprogramming of the transcriptome.

Oil Penetration of Batter-Breaded Fish Nuggets during Deep-Fat Frying: Effect of Frying Oils.

Four frying oils (rapeseed, soybean, rice bran, and palm oils) were employed either as received (fresh) or after preheating at 180 °C for 10 h, and measured for their fatty acid composition, viscosity, and dielectric constant. Batter-breaded fish nuggets (BBFNs) were fried at 180 °C (60 s), and the effect of the oils' quality on the oil penetration of fried BBFNs were investigated via the analysis of the absorption and the distribution of fat. Preheating increased the viscosity and dielectric constant of the oils. The total fat content using fresh oils was the greatest for palm oil (14.2%), followed by rice bran oil (12.2%), rapeseed oil (12.1%), and soybean oil (11.3%), a trend that was nearly consistent with the penetrated surface oil, except that the penetrated oil for soybean oil (6.8%) was higher than rapeseed oil (6.3%). The BBFNs which were fried using fresh oils possessed a more compact crust and smaller pores for the core and underwent a lower oil penetration compared to the preheated oils. The results suggested that the oils' quality significantly affected the oil penetration of fried BBFNs.

RNA-Seq analysis and de novo transcriptome assembly of Hevea brasiliensis.

Hevea brasiliensis, being the only source of commercial natural rubber, is an extremely economically important crop. In an effort to facilitate biological, biochemical and molecular research in rubber biosynthesis, here we report the use of next-generation massively parallel sequencing technologies and de novo transcriptome assembly to gain a comprehensive overview of the H. brasiliensis transcriptome. The sequencing output generated more than 12 million reads with an average length of 90 nt. In total 48,768 unigenes (mean size = 436 bp, median size = 328 bp) were assembled through de novo transcriptome assembly. Out of 13,807 H. brasiliensis cDNA sequences deposited in Genbank of the National Center for Biotechnology Information (NCBI) (as of Feb 2011), 11,746 sequences (84.5%) could be matched with the assembled unigenes through nucleotide BLAST. The assembled sequences were annotated with gene descriptions, Gene Ontology (GO) and Clusters of Orthologous Group (COG) terms. In all, 37,432 unigenes were successfully annotated, of which 24,545 (65.5%) aligned to Ricinus communis proteins. Furthermore, the annotated uingenes were functionally classified according to the GO, COG and Kyoto Encyclopedia of Genes and Genomes databases. Our data provides the most comprehensive sequence resource available for the study of rubber trees as well as demonstrates effective use of Illumina sequencing and de novo transcriptome assembly in a species lacking genomic information.

Identification of JAZ-interacting MYC transcription factors involved in latex drainage in Hevea brasiliensis.

Hevea brasiliensis Müll. Arg. is one of the most frequently wounded plants worldwide. Expelling latex upon mechanical injury is a wound response of rubber trees. However, JA-mediated wound responses in rubber trees are not well documented. In this work, three JAZ-interacting MYC transcription factors of H. brasiliensis (termed HbMYC2/3/4) were identified by yeast two-hybrid screening. HbMYC2/3/4 each showed specific interaction profiles with HbJAZs. HbMYC2/3/4 each localized in the nucleus and exhibited strong transcriptional activity. To identify the target genes potentially regulated by HbMYC2/3/4, cis-elements interacting with HbMYC2/3/4 were first screened by yeast one-hybrid assays; the results indicated that HbMYC2/3/4 each could bind G-box elements. Additional analysis confirmed that HbMYC2/3/4 bound the HbPIP2;1 promoter, which contains five G-box cis-elements, and regulated the expression of reporter genes in yeast cells and in planta. HbMYC2/3/4 were induced by exogenous JA treatment but suppressed by ethylene (ET) treatment; in contrast, HbPIP2;1 was positively regulated by ET but negatively regulated by JA treatment. Given that HbPIP2;1 is involved in latex drainage, it could be proposed that HbMYC2/3/4 are involved in the regulation of HbPIP2;1 expression as well as latex drainage, both of which are coordinated by the JA and ET signalling pathways.