Tapping panel dryness (TPD) occurrence in high latex yielding rubber tree (Hevea brasiliensis) is characterized by the partial or complete cessation of latex flow upon tapping leading to severe loss in natural rubber production around the world. The goal of this study was to identify genes whose mRNA transcript levels are differentially regulated in rubber tree during the onset of TPD. To isolate TPD responsive genes, two cDNA libraries (forward and reverse) from total RNA isolated from latex of healthy and TPD trees were constructed using suppression subtractive hybridization (SSH) method. In total, 1,079 EST clones were obtained from two cDNA libraries and screened by reverse Northern blot analysis. Screening results revealed that about 352 clones were differentially regulated and they were selected for sequencing. Based on the nucleotide sequence data, the putative functions of cDNA clones were predicted by BLASTX/BLASTN analysis. Among these, 64 were genes whose function had been previously identified while the remaining clones were genes with either unknown protein function or insignificant similarity to other protein/DNA/EST sequences in existing databases. RT-PCR analysis was carried out to validate the up-regulated genes from both the libraries. Among them, two genes were strongly down-regulated in TPD trees. The level of mRNA transcripts of these two genes was further examined by conventional Northern and RT-PCR analysis. Results indicated that the expression level of two genes was significantly lower in TPD trees compared to healthy trees. Many TPD associated genes were also up-regulated in TPD trees suggesting that they may be involved in triggering programmed cell death (PCD) during the onset of TPD syndrome. The results presented here demonstrate that SSH technique provides a powerful complementary approach for the identification of TPD related genes from rubber tree.

In rubber tree (Hevea brasiliensis), tapping panel dryness (TPD) syndrome is considered as a complex physiological disorder which affects latex biosynthesis. To identify differentially expressed genes between healthy and TPD-affected trees, mRNA differential display reverse transcriptase polymerase chain reaction (DDRT-PCR) analysis was performed. We isolated 10 differentially expressed cDNA fragments of which one cDNA encoding a putative TOM20 like protein was identified. The cDNA (1,024 bp), corresponding to the HbTOM20 gene (H evea b rasiliensis Translocase of the Outer Mitochondrial Membrane), contained an open reading frame to code for 202 amino acid protein with a theoretical pI value of 9.5 and the calculated protein M (W) was 23.5 kDa. The predicted amino acid sequence contained conserved domains of TOM20 like proteins in the N-terminal. The protein HbTOM20 has 32% and 27% similarity to Populus TOM20 and Solanum TOM20, respectively. Both semi-quantitative RT-PCR and Northern blot results revealed that the HbTOM20 expression was significantly down-regulated in TPD-affected trees compared to healthy one. Accumulation of HbTOM20 mRNA transcripts was significantly higher in the bark tissues collected from healthy region than that of partially affected by TPD (partially dried) while barely detectable in completely TPD-affected area. Differential expression pattern was noticed in three rubber clones representing various degrees of TPD tolerance. These results suggest that down-regulation of HbTOM20 in TPD-affected trees may play an important role in alteration of mitochondrial metabolism resulting in impaired latex biosynthesis.

In Hevea tree, rubber elongation factor (REF) is a key gene involved in rubber biosynthesis. Since the immaturity period for Hevea is 6 years, identification of molecular marker for latex yield potential will be beneficial for early selection of high yielding clones. The main objective of this research is to study the expression pattern of the REF gene in contrasting latex yield rubber clones (high and low yielding) by Northern blot as well as RT-PCR analysis. Accumulation of REF mRNA transcripts was significantly higher in latex cells compared to other cells of seedlings and mature Hevea trees. Northern results revealed that the level of REF gene expression in latex cells of high yielding rubber clones was significantly higher than in low yielders. According to RT-PCR results, the abundance of REF mRNA transcripts in latex cells was fivefold higher in the RRII105 clone, one of the most high yielding rubber clones. It is evident from the results that both tapping and ethephon treatment had a direct effect on induction of REF gene expression. Results demonstrate a positive correlation between REF gene expression pattern and latex yield.

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Most cultivated rubber tree (Hevea brasiliensis Willd. ex A. Juss.) clones in India are susceptible to abnormal leaf fall disease (ALF), which is caused by various Phytophthora species and results in yield losses of up to 40%. Because the conventional breeding programs for this perennial tree crop are complex and time consuming, we attempted to find a molecular solution to increase the tolerance of rubber trees to ALF. The expression patterns of the gene coding for the pathogenesis-related beta-1,3-glucanase (beta-glu) enzyme in a tolerant (RRII 105) and a highly susceptible (RRIM 600) clone of rubber tree were examined, following infection with ALF-causing Phytophthora meadii McRae. Infected leaf samples were collected at different times after inoculation, and RNA was extracted and subjected to Northern blot hybridization and reverse transcriptase polymerase chain reaction (RT-PCR). On hybridization with a 1.25 kb beta-glu probe, Northern blots showed a marked increase in beta-glu transcript levels in both clones 48 h after inoculation. However, compared with the susceptible RRIM 600 clone, the tolerant RRII 105 clone had a higher rate of increase and a more prolonged induction, with beta-glu transcript levels remaining high for 4 days after inoculation. In RRIM 600, the mRNA levels decreased significantly 48 h after inoculation. On re-hybridization with an 18S rRNA probe, uniform signals were detected in all the lanes, indicating that an equal amount of total RNA was present in all samples. Similar results were obtained in relative quantitative RT-PCR experiments with the housekeeping actin gene as an internal control. Thus, although induction of the beta-glu gene occurred in both tolerant and susceptible clones, the predominant difference between clones was in the intensity and duration of the response. The tolerance of clone RRII 105 may be associated with the prolonged expression of the gene following infection. The antifungal activity of these hydrolase enzymes makes them rational candidates for overexpression by genetic transformation to produce disease resistant crops.