Revealing the dominant long noncoding RNAs responding to the infection with Colletotrichum gloeosporioides in Hevea brasiliensis.

BACKGROUND: Rubber tree (Hevea brasiliensis) acts as an important tropic economic crop and rubber tree anthracnose, mainly caused by Colletotrichum gloeosporioides, is one of the most common fungal disease, which leads to serious loss of rubber production. Therefore, the investigation on disease resistance is of great worldwide significance. In the past decades, substantial progress has been made on coding gene families related with plant disease resistance. However, in rubber tree, whether the disease resistance mechanism involves noncoding RNAs, especially long noncoding RNAs (lncRNAs), still remains poorly understood. RESULTS: Here, we modeled the development of H. brasiliensis leaf samples inoculated with C. gloeosporioides at divergent stages, explored to identify the expressed ncRNAs by RNA-seq, and investigated the dominant lncRNAs responding to the infection, through constructing a co-expressed network systematically. On the dominant lncRNAs, we explored the potential functional role of lncRNA11254 recruiting the transcription factor, and that lncRNA11041 and lncRNA11205 probably stimulate the accumulation of corresponding disease responsive miRNAs, and further modulate the expressions of target genes, accompanying with experimental examination. CONCLUSIONS: Take together, computational analyses in silico and experimental evidences in our research collectively revealed the responsive roles of dominant lncRNAs to the pathogen. The results will provide new perspectives to unveil the plant disease resistance mechanisms, and will presumably provide a new theoretical basis and candidate prognostic markers for the optimization and innovation of genetic breeding for rubber tree. REVIEWERS: This article was reviewed by Ryan McGinty and Roland Huber.

Proteomics analysis of latex from Hevea brasiliensis (clone RRIM 600).

The natural rubber latex extracted from the bark of Hevea brasiliensis plays various important roles in today's modern society. Following ultracentrifugation, the latex can be separated into 3 layers: C-serum, lutoids, and rubber particles. Previous studies have shown that a large number of proteins are present in these 3 layers. However, a complete proteome for this important plant is still unavailable. Protein sequences have been recently translated from the completed draft genome database of H. brasiliensis, leading to the creation of annotated protein databases of the following H. brasiliensis biosynthetic pathways: photosynthesis, latex allergens, rubberwood formation, latex biosynthesis, and disease resistance. This research was conducted to identify the proteins contained within the latex by way of de novo sequencing from mass spectral data obtained from the 3 layers of the latex. Peptides from these proteins were fragmented using collision-induced dissociation, higher-energy collisional dissociation, and electron-transfer dissociation activation methods. A large percentage of proteins from the biosynthetic pathways (63% to 100%) were successfully identified. In addition, a total of 1839 unique proteins were identified from the whole translated draft genome database (AnnHBM).

Identification of Powdery Mildew Responsive Genes in Hevea brasiliensis through mRNA Differential Display.

Powdery mildew is an important disease of rubber trees caused by Oidium heveae B. A. Steinmann. As far as we know, none of the resistance genes related to powdery mildew have been isolated from the rubber tree. There is little information available at the molecular level regarding how a rubber tree develops defense mechanisms against this pathogen. We have studied rubber tree mRNA transcripts from the resistant RRIC52 cultivar by differential display analysis. Leaves inoculated with the spores of O. heveae were collected from 0 to 120 hpi in order to identify pathogen-regulated genes at different infection stages. We identified 78 rubber tree genes that were differentially expressed during the plant-pathogen interaction. BLAST analysis for these 78 ESTs classified them into seven functional groups: cell wall and membrane pathways, transcription factor and regulatory proteins, transporters, signal transduction, phytoalexin biosynthesis, other metabolism functions, and unknown functions. The gene expression for eight of these genes was validated by qRT-PCR in both RRIC52 and the partially susceptible Reyan 7-33-97 cultivars, revealing the similar or differential changes of gene expressions between these two cultivars. This study has improved our overall understanding of the molecular mechanisms of rubber tree resistance to powdery mildew.

Structural and phylogenetic analysis of Pto-type disease resistance gene candidates in Hevea brasiliensis.

The tomato Pto gene encodes a serine/threonine kinase (STK) whose molecular characterization has provided valuable insights into the disease resistance mechanism of tomato. Therefore, Pto is considered as a promising candidate for engineering broad-spectrum pathogen resistance in this crop. In this study, a pair of degenerate primers based on conserved subdomains of plant STKs similar to the tomato Pto protein was used to amplify similar sequences in a hevea cultivar (Hevea brasiliensis Muell. Arg). A fragment of ~550 bp was amplified, cloned, and sequenced. The sequence analysis of several clones revealed 12 distinct sequences highly similar to STKs. Based on their significant similarity with the tomato Pto protein (BLASTX E value<3e-53), seven sequences were classified as Pto resistance gene candidates (Pto-RGCs). Multiple sequence alignment of the hevea Pto-RGC products revealed that these sequences contain several conserved subdomains present in most STKs, as well as several conserved residues that are crucial for Pto function. Moreover, phylogenetic analysis showed that the hevea Pto-RGCs clustered with Pto, suggesting a common evolutionary origin with this resistance gene. The Pto-RGCs isolated in this study represent a valuable sequence resource that could assist in the development of disease resistance in hevea.

In-depth proteome analysis of the rubber particle of Hevea brasiliensis (para rubber tree).

The rubber particle is a special organelle in which natural rubber is synthesised and stored in the laticifers of Hevea brasiliensis. To better understand the biological functions of rubber particles and to identify the candidate rubber biosynthesis-related proteins, a comprehensive proteome analysis was performed on H. brasiliensis rubber particles using shotgun tandem mass spectrometry profiling approaches-resulting in a thorough report on the rubber particle proteins. A total of 186 rubber particle proteins were identified, with a range in relative molecular mass of 3.9-194.2 kDa and in isoelectric point values of 4.0-11.2. The rubber particle proteins were analysed for gene ontology and could be categorised into eight major groups according to their functions: including rubber biosynthesis, stress- or defence-related responses, protein processing and folding, signal transduction and cellular transport. In addition to well-known rubber biosynthesis-related proteins such as rubber elongation factor (REF), small rubber particle protein (SRPP) and cis-prenyl transferase (CPT), many proteins were firstly identified to be on the rubber particles, including cyclophilin, phospholipase D, cytochrome P450, small GTP-binding protein, clathrin, eukaryotic translation initiation factor, annexin, ABC transporter, translationally controlled tumour protein, ubiquitin-conjugating enzymes, and several homologues of REF, SRPP and CPT. A procedure of multiple reaction monitoring was established for further protein validation. This comprehensive proteome data of rubber particles would facilitate investigation into molecular mechanisms of biogenesis, self-homeostasis and rubber biosynthesis of the rubber particle, and might serve as valuable biomarkers in molecular breeding studies of H. brasiliensis and other alternative rubber-producing species.

Draft genome sequence of the rubber tree Hevea brasiliensis.

BACKGROUND: Hevea brasiliensis, a member of the Euphorbiaceae family, is the major commercial source of natural rubber (NR). NR is a latex polymer with high elasticity, flexibility, and resilience that has played a critical role in the world economy since 1876. RESULTS: Here, we report the draft genome sequence of H. brasiliensis. The assembly spans ~1.1 Gb of the estimated 2.15 Gb haploid genome. Overall, ~78% of the genome was identified as repetitive DNA. Gene prediction shows 68,955 gene models, of which 12.7% are unique to Hevea. Most of the key genes associated with rubber biosynthesis, rubberwood formation, disease resistance, and allergenicity have been identified. CONCLUSIONS: The knowledge gained from this genome sequence will aid in the future development of high-yielding clones to keep up with the ever increasing need for natural rubber.

Natural rubber biosynthesis in plants: rubber transferase.

Rubber biosynthesis in plants is a fascinating biochemical system, which evolved at the dawn of the dicotyledoneae and is present in at least four of the dictolydonous superorders. Rubber biosynthesis is catalyzed by a membrane complex in a monolayer membrane envelope, requires two distinct substrates and a divalent cation cofactor, and produces a high-molecular-weight isoprenoid polymer. A solid understanding of this system underpins valuable papers in the literature. However, the published literature is rife with unreliable reports in which the investigators have fallen into traps created by the current incomplete understanding of the biochemistry of rubber synthesis. In this chapter, we attempt to guide both new and more established researchers around these pitfalls.

The clinical meaning of positive latex sIgE in patients with food/pollen adverse reactions.

Natural rubber latex allergy (NRL-A) is an international problem of public health. About 50-60% of NRL-A patients may present adverse reactions after ingestion of cross-reacting vegetable foods. This condition, called "Latex-fruit Syndrome", is a matter of research. The aim of our study is to distinguish between clinical/subclinical latex-fruit syndrome and cross-sensitization to latex and food/pollen allergens on the basis of latex recombinant allergens. We studied 51 patients with food hypersensitivity and serological evidence of NRL sensitization. The subjects underwent an accurate allergological evaluation (skin prick test with latex, food and pollen extracts, specific IgE to latex and recombinant allergens, challenge provocation tests). The patients were divided in two groups: group A) 34 patients with clinical and serological latex and fruit/vegetable allergies; group B) 17 patients allergic to fruits/vegetables and/or pollens, with serological, but not clinical NRL-A. All the latex challenge tests resulted positive in group A patients and only two patients of group B presented positive cutaneous challenge tests. Moreover, specific IgE-antibodies were detected to rHev b 5, to rHev b 6.01, to rHev b 6.02 and to rHev b 8 (and other profilins) of group A patients, while in group B we observed a monosensitization to Hev b8, probably linked to a cross-sensitization to pollens and foods. At the present state of knowledge, we need a multi-parametric approach based on a combination of clinical history, diagnostic tests (CRD) and latex challenge tests to make diagnosis of latex-fruit syndrome.

Diagnosis of latex allergy: the importance of hev B 11.

We present the cases of 5 patients with a positive clinical history of cutaneous symptoms due to contact with latex products. A latex allergological assessment was made through skin prick tests (SPTs) both with commercial latex extracts and extemporaneous glove extracts, and serum-specific IgE to latex and glove-use tests. In addition, serum-specific IgE to recombinant allergens for Hevea brasiliensis was dosed. Molecular diagnostics in association with the glove-use test and, to a lesser extent, the SPTs with glove eluate are useful diagnostic tests to confirm the diagnosis of latex allergy in patients with mucocutaneous symptoms.

Defense-related polyphenol oxidase from Hevea brasiliensis cell suspension: purification and characterization.

Polyphenol oxidase (PPO) was examined from the extract of leaf, seed, and cell suspension of Hevea brasiliensis, a rubber plant. The defense-related isozyme from Hevea cell suspension induced by culture filtrate of Phytophthora palmivora or by agitation stress was isolated through anion exchange and affinity chromatography, respectively. A 104-purification fold, migrated as a single band of 70 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis of PPO, was obtained after further purified by the preparative gel electrophoresis. Based on reaction with catechol and dopamine but not with p-cresol and guaiacol, it is a diphenol-type PPO. The values of V(max)/K(m) ratio indicated that catechol was the most specific substrate. The optimal activity of the purified PPO was observed at pH 6.0. The PPO activity was retained at pH 4.0-10.0 and temperature 10-60 °C. The inhibitors which completely inhibited the activity were ascorbic acid, dithiothreitol, and ß-mercaptoethanol while sodium azide was a poor inhibitor. The PPO obtained from Hevea cell suspension possesses high specific activity and is stable at wide range of pH and temperature. It is therefore suitable for extreme condition uses and may lead to an alternative source of PPO in various industrial applications.

Primer reporte de susceptibilidad del clon de caucho natural FX-3864 a Microcyclus ulei en la altillanura colombiana / First report of susceptibility of natural rubber clone FX-3864 a Microcyclus ulei en la altillanuera colombiana

El mal suramericano de las hojas (SALB), enfermedad endémica del caucho (Hevea brasiliensis), es causado por Microcyclus ulei (forma imperfecta Fusicladium macrosporum) y constituye el principal limitante del cultivo en América, área donde el microorganismo patógeno es endémico. En forma semejante al de otros cultivos agrícolas, el manejo de esta enfermedad está condicionado a la disponibilidad de resistencia genética en el hospedero. En razón de su productividad y condición de resistencia genética, el clon FX 3864 ha sido ampliamente plantado en zonas con diferente potencialidad epidémica a la incidencia del SALB en Colombia, particularmente las denominadas de “no escape” a la enfermedad. Durante el 2010, plantaciones con el clon FX 3864 en fase productiva presentaron síntomas de SALB en zonas de escape ubicadas en la altillanura colombiana (departamento del Meta). En parcelas trampa ubicadas en áreas aledañas a los cultivos se estableció que la severidad promedio de la enfermedad alcanzó niveles de 5,78% en este clon. Verificada la causalidad de la enfermedad mediante observaciones al microscopio se procedió a confirmar el origen del material sobre el cual se desarrollaban las lesiones, utilizando marcadores moleculares (4 microsatélites específicos). Los resultados de la prueba permitieron confirmar la susceptibilidad del hasta hace poco resistente clon FX 3864 al SALB en Colombia. Se sugiere tomar en consideración la nueva condición de este clon y, en concordancia, reorientar los programas de fomento del cultivo advirtiendo a los agricultores sobre los riesgos potenciales de ocurrencia de la enfermedad en las nuevas áreas programadas.

South American Leaf Blight (SALB), caused by Microcyclus ulei (anamorph Fusicladium macrosporum), is an endemic major disease of the rubber tree (Hevea brasiliensis) in America. As well as in other crop systems, its management on rubber plantations relies on plant genetic resistance availability, among other means. FX 3864 is a rubber tree clone widely planted in Colombia due to its production capability and disease resistance. During 2010 SALB symptoms developed in commercial crops at the Meta region of Colombia. Crop traps located nearby the plantations showed mean disease severity levels of 5.78%. Once the causal organism was microscopically confirmed as responsible for the diseased tissue, their origin was characterized by molecular means using 4 microsatellites specific to the rubber tree. The procedure confirmed that FX 3864 was the clone of origin of the leaf tissue. SALB occurring over FX 3864 implies the need to redirect crop disease management measures to be followed on the new development areas of rubber cultivation, warning growers about potential hazards of disease incidence.

A proteomic investigation of B lymphocytes in an autistic family: a pilot study of exposure to natural rubber latex (NRL) may lead to autism.

Autism is a multi-factorial neurodevelopmental disorder. We have investigated the molecular mechanism involved in a Chinese family with autism by a proteomic approach. Antibody chips containing 500 spots of human protein antibodies were used to screen for differentially expressed proteins in the peripheral B lymphocytes between autistic and non-autistic siblings in this family. Four proteins relevant to immuno-pathway, including IKKα that was up-regulated and Tyk2, EIF4G1 and PRKCI that were down-regulated, were identified differentially expressed in autistic versus non-autistic siblings. Western blot analysis and reverse transcription quantitative polymerase chain reaction validated the differential expression of these four proteins. Based on the function of these differentially expressed proteins, relevant studies on immunoglobulin E (IgE) level, nuclear factor kappa B signaling activation and cell cycle were conducted in both autistic and non-autistic children of this family. Considering the fact that the family members were in close contact with natural rubber latex (NRL) and that IgE-mediated cross-reactions could be triggered by Hevea brasiliensis (Hev-b) proteins in NRL, we hypothesize that immune reactions triggered by close contact with NRL might influence the functions of B lymphocytes by altering expression of certain proteins identified in our experiments thus contributing to the occurrence of autism.

Identification of clinically relevant cross-sensitization between Soliadgo virgaurea (goldenrod) and Hevea brasiliensis (natural rubber latex).

BACKGROUND: Solidago virgaurea (goldenrod) is a perennial weed from which no allergens have been identified. A high latex content in its leaves has been reported. Although not an airborne allergen, it may be an important occupational sensitizer. OBJECTIVE: To identify allergenic proteins in goldenrod and to determine whether they cross-react with Hevea brasiliensis latex. METHODS: Potential cross-reactive allergens in latex and goldenrod were investigated by immunoblot inhibition and ImmunoCAP inhibition analyses using serum from patients with clinically evident goldenrod and/or latex allergy. Cross reactivity between latex allergens and goldenrod proteins was studied using recombinant Hev b 1, 3, 4, 5, 6.01, 6.02, 8, 9, or 11 in ImmunoCAP inhibition analyses. RESULTS: Immunoglobulin (Ig) E antibodies from individuals with goldenrod allergy bound extracted goldenrod proteins ranging from 20 kDa to 130 kDa in Western blots. Evidence for latex and goldenrod cross reactivity was identified by ImmunoCAP and immunoblot inhibition experiments using serum from patients with strongly positive concomitant latex and goldenrod-specific IgE antibody responses. Observed latex-goldenrod cross reactivity could not be ascribed to any of the recombinant major latex allergens evaluated. CONCLUSIONS: H brasiliensis latex and goldenrod contain cross-reactive and unique allergenic proteins. Exposure to goldenrod may sensitize patients to latex and vice versa.

Microarrays of recombinant Hevea brasiliensis proteins: a novel tool for the component-resolved diagnosis of natural rubber latex allergy.

BACKGROUND: Component-resolved diagnosis using microarray technology has recently been introduced in clinical allergology, but its applicability in patients with natural rubber latex (NRL) allergy has not been investigated. OBJECTIVES: To evaluate the utility of microarray-based immunoglobulin (Ig) E detection in the diagnostic workup of NRL allergy and to compare this new diagnostic tool with established methods of NRL-specific IgE detection. METHODS: We investigated 52 adults with immediate-type NRL allergy and 50 control patients. Determination of specific serum IgE against 8 recombinant Hevea brasiliensis allergen components was performed using a customized allergen microarray and a conventional fluorescence enzyme immunoassay (FEIA). RESULTS: The panel of microarrayed allergen components was shown to represent a comprehensive repertoire of clinically relevant NRL proteins. NRL-specific IgE recognition patterns and sensitization rates determined by microarray analysis were similar to those obtained by conventional FEIA. The diagnostic sensitivity rates of combined single-component data were not significantly different for the respective recombinant test system, whereas the sensitivity level of extract-based FEIA analysis was markedly higher. CONCLUSION: The current study provides evidence that microarrays of recombinant NRL allergen components are a suitable new tool for the diagnosis of NRL-specific sensitization.They show performance characteristics comparable to those of current diagnostic tests and could be indicated in small children in whom only limited blood volumes are obtainable. Further large-scale studies in unselected patient populations and in high-risk groups are warranted before the microarray can be introduced into routine management of patients with NRL allergy.

In-depth exploration of Hevea brasiliensis latex proteome and "hidden allergens" via combinatorial peptide ligand libraries.

The proteome of Hevea brasiliensis latex has been explored in depth via combinatorial peptide ligand libraries. A total of 300 unique gene products have been identified in this latex, whose proteome has been largely unknown up to the present. In search for unknown allergens, control latex and eluates from the ligand libraries have been fractionated by two-dimensional mapping, blotted and confronted with sera of 18 patients. In addition to the already known and named Hevea major allergens, we have unambiguously detected several others like, for instance: heat shock protein (81 kDa), proteasome subunit (30 kDa), protease inhibitor (8 kDa), hevamine A (43 kDa) and glyceraldehyde-3-phosphate dehydrogenase (37 kDa). Gene Ontology analysis of analyzed fractions has shown that major functions are substantially unchanged after sample treatment, while novel biological functions appeared that were undetectable in the crude sample.

Patients monosensitised to Hev b 8 (Hevea brasiliensis latex profilin) may safely undergo major surgery in a normal (non-latex safe) environment.

BACKGROUND: Natural rubber latex allergy is a condition at high risk of anaphylaxis during surgery. However, latex contains several protein allergens and not all of them may show the same clinical relevance. OBJECTIVE: To investigate the clinical relevance of Hey b 8, the natural rubber latex profilin. METHODS: Seven patients without a clinical history of latex allergy but identified as being latex hypersensitive by positive SPT (3/7) and or positive latex-specific IgE during routine pre-surgery allergy investigations were studied. All patients were monosensitized to Hev b 8 (Hevea brasiliensis latex profilin) as shown by the detection of specific IgE to recombinant latex allergen components. Ten subjects with a history of latex allergy (urticaria, asthma, and/or rhinitis), sensitised to latex allergens other than profilin were enrolled as controls. Both patients and controls underwent a latex glove-wearing test; in case of a negative test, patients underwent surgery in a normal surgical setting. RESULTS: All 7 patients scored negative on latex glove wearing test and underwent major surgery (orthopaedic, Caesarean section, pilonidal sinus, vascular, tonsillectomy, uterine revision, and uretral surgery) in a normal (non-latex safe) surgical setting without any consequence. In contrast, 9/10 (90%) controls showed a positive latex glove-wearing test (p < 0.01). CONCLUSION: Latex profilin is either clinically irrelevant or is no longer present in latex products. This study highlights the importance of a component-resolved diagnosis of latex sensitisation as a tool to get a more precise assessment of the risk and to reduce the costs of healthcare.