Endophytes from Wild Rubber Trees as Antagonists of the Pathogen Corynespora cassiicola.

The Corynespora leaf fall disease of rubber trees, caused by the necrotrophic fungus Corynespora cassiicola, is responsible for important yield losses in Asian and African plantations, whereas its impact is negligible in South America. The objective of this study was to identify potential antagonists of C. cassiicola among fungal endophytes (i.e., Pestalotiopsis, Colletotrichum, and Trichoderma spp.) isolated from wild and cultivated rubber trees distributed in the Peruvian Amazon. We first tested the endophytes in dual in vitro confrontation assays against a virulent C. cassiicola isolate (CCP) obtained from diseased rubber trees in the Philippines. All Trichoderma isolates overran the CCP colony, suggesting some antagonistic mechanism, while species from the other genera behaved as mutual antagonists. Trichoderma isolates were then tested through antibiosis assays for their capacity to produce growth-inhibiting molecules. One isolate (LA279), recovered as an endophyte from a wild Hevea guianensis specimen and identified as Trichoderma koningiopsis, showed significant antibiosis capacity. We demonstrated that LA279 was also able to endophytically colonize the cultivated rubber tree species (H. brasiliensis). Under controlled laboratory conditions, rubber plants were inoculated with three Trichoderma strains, including LA279, in combination with the pathogenic CCP. Results showed that 1 week preinoculation with the endophytes differentially reduced CCP mycelial development and symptoms. In conclusion, this study suggests that T. koningiopsis isolate LA279-and derivate compounds-could be a promising candidate for the biological control of the important rubber tree pathogen C. cassiicola.

Overexpression of Hevea brasiliensis ethylene response factor HbERF-IXc5 enhances growth and tolerance to abiotic stress and affects laticifer differentiation.

Ethylene response factor 1 (ERF1) is an essential integrator of the jasmonate and ethylene signalling pathways coordinating a large number of genes involved in plant defences. Its orthologue in Hevea brasiliensis, HbERF-IXc5, has been assumed to play a major role in laticifer metabolism and tolerance to harvesting stress for better latex production. This study sets out to establish and characterize rubber transgenic lines overexpressing HbERF-IXc5. Overexpression of HbERF-IXc5 dramatically enhanced plant growth and enabled plants to maintain some ecophysiological parameters in response to abiotic stress such as water deficit, cold and salt treatments. This study revealed that HbERF-IXc5 has rubber-specific functions compared to Arabidopsis ERF1 as transgenic plants overexpressing HbERF-IXc5 accumulated more starch and differentiated more latex cells at the histological level. The role of HbERF-IXc5 in driving the expression of some target genes involved in laticifer differentiation is discussed.

Ethylene Response Factors Are Controlled by Multiple Harvesting Stresses in Hevea brasiliensis.

Tolerance of recurrent mechanical wounding and exogenous ethylene is a feature of the rubber tree. Latex harvesting involves tapping of the tree bark and ethephon is applied to increase latex flow. Ethylene is an essential element in controlling latex production. The ethylene signalling pathway leads to the activation of Ethylene Response Factor (ERF) transcription factors. This family has been identified in Hevea brasiliensis. This study set out to understand the regulation of ERF genes during latex harvesting in relation to abiotic stress and hormonal treatments. Analyses of the relative transcript abundance were carried out for 35 HbERF genes in latex, in bark from mature trees and in leaves from juvenile plants under multiple abiotic stresses. Twenty-one HbERF genes were regulated by harvesting stress in laticifers, revealing an overrepresentation of genes in group IX. Transcripts of three HbERF-IX genes from HbERF-IXc4, HbERF-IXc5 and HbERF-IXc6 were dramatically accumulated by combining wounding, methyl jasmonate and ethylene treatments. When an ethylene inhibitor was used, the transcript accumulation for these three genes was halted, showing ethylene-dependent induction. Subcellular localization and transactivation experiments confirmed that several members of HbERF-IX are activator-type transcription factors. This study suggested that latex harvesting induces mechanisms developed for the response to abiotic stress. These mechanisms probably depend on various hormonal signalling pathways. Several members of HbERF-IX could be essential integrators of complex hormonal signalling pathways in Hevea.

Transcriptional and post-transcriptional regulation of the jasmonate signalling pathway in response to abiotic and harvesting stress in Hevea brasiliensis.

BACKGROUND: Latex harvesting in Hevea brasiliensis amounts to strong abiotic stress that can cause a halt in production in the most susceptible clones. Although the role of jasmonic acid has been suggested in laticifer differentiation, its role in latex production and in the response to harvesting stress has received very little attention. Only a few key genes acting in the COI-JAZ-MYC module have been isolated and studied at transcriptional level. RESULTS: Use of a reference transcriptome obtained on rubber clone PB 260 covering a large number of tissues under different environmental conditions enabled us to identify 24 contigs implicated in the jasmonate signalling pathway in the rubber tree. An analysis of their expression profile by qPCR, combined with hierarchical clustering, suggested that the jasmonate signalling pathway is highly activated in laticifer cells and, more particularly, in the response to harvesting stress. By comparison with their genomic sequences, the existence of regulation by alternative splicing was discovered for JAZ transcripts in response to harvesting stress. Lastly, positive transcriptional regulation of the HbJAZ_1405 gene by MYC was demonstrated. CONCLUSION: This study led to the identification of all actors of jasmonate signalling pathway and revealed a specific gene expression pattern in latex cells. In-depth analysis of this regulation showed alternative splicing that has been previously shown in Arabidopsis. Interestingly, genotypic variation was observed in Hevea clones with contrasting latex metabolism. This result suggests an involvement of jasmonate signalling pathway in latex production. The data suggest that specific variability of the JA pathway may have some major consequences for resistance to stress. The data support the hypothesis that a better understanding of transcriptional regulations of jasmonate pathway during harvesting stress, along with the use of genotypic diversity in response to such stress, can be used to improve resistance to stress and rubber production in Hevea.

Sequence and expression analyses of ethylene response factors highly expressed in latex cells from Hevea brasiliensis.

The AP2/ERF superfamily encodes transcription factors that play a key role in plant development and responses to abiotic and biotic stress. In Hevea brasiliensis, ERF genes have been identified by RNA sequencing. This study set out to validate the number of HbERF genes, and identify ERF genes involved in the regulation of latex cell metabolism. A comprehensive Hevea transcriptome was improved using additional RNA reads from reproductive tissues. Newly assembled contigs were annotated in the Gene Ontology database and were assigned to 3 main categories. The AP2/ERF superfamily is the third most represented compared with other transcription factor families. A comparison with genomic scaffolds led to an estimation of 114 AP2/ERF genes and 1 soloist in Hevea brasiliensis. Based on a phylogenetic analysis, functions were predicted for 26 HbERF genes. A relative transcript abundance analysis was performed by real-time RT-PCR in various tissues. Transcripts of ERFs from group I and VIII were very abundant in all tissues while those of group VII were highly accumulated in latex cells. Seven of the thirty-five ERF expression marker genes were highly expressed in latex. Subcellular localization and transactivation analyses suggested that HbERF-VII candidate genes encoded functional transcription factors.

Some ethylene biosynthesis and AP2/ERF genes reveal a specific pattern of expression during somatic embryogenesis in Hevea brasiliensis.

BACKGROUND: Ethylene production and signalling play an important role in somatic embryogenesis, especially for species that are recalcitrant in in vitro culture. The AP2/ERF superfamily has been identified and classified in Hevea brasiliensis. This superfamily includes the ERFs involved in response to ethylene. The relative transcript abundance of ethylene biosynthesis genes and of AP2/ERF genes was analysed during somatic embryogenesis for callus lines with different regeneration potential, in order to identify genes regulated during that process. RESULTS: The analysis of relative transcript abundance was carried out by real-time RT-PCR for 142 genes. The transcripts of ERFs from group I, VII and VIII were abundant at all stages of the somatic embryogenesis process. Forty genetic expression markers for callus regeneration capacity were identified. Fourteen markers were found for proliferating calli and 35 markers for calli at the end of the embryogenesis induction phase. Sixteen markers discriminated between normal and abnormal embryos and, lastly, there were 36 markers of conversion into plantlets. A phylogenetic analysis comparing the sequences of the AP2 domains of Hevea and Arabidopsis genes enabled us to predict the function of 13 expression marker genes. CONCLUSIONS: This first characterization of the AP2/ERF superfamily in Hevea revealed dramatic regulation of the expression of AP2/ERF genes during the somatic embryogenesis process. The gene expression markers of proliferating callus capacity to regenerate plants by somatic embryogenesis should make it possible to predict callus lines suitable to be used for multiplication. Further functional characterization of these markers opens up prospects for discovering specific AP2/ERF functions in the Hevea species for which somatic embryogenesis is difficult.

Variation in GUS activity in vegetatively propagated Hevea brasiliensis transgenic plants.

Hevea brasiliensis transgenic plants are regenerated from transgenic callus lines by somatic embryogenesis. Somatic embryogenesis is not yet available for commercial propagation of Hevea clones, which requires conventional grafting of buds on rootstock seedlings (budding). The stability of transgene expression in budded plants is therefore necessary for further development of genetic engineering in rubber trees. Transgene expression was assessed by fluorimetric beta-glucuronidase (GUS) activity in fully developed leaves of in vitro plants from transgenic lines and their sub-lines obtained by budding. A large variation in GUS activity was found in self-rooted in vitro plants of five transgenic lines, and the absence of activity in one line suggested transgene silencing. Beyond confirming transmissibility of the reporter gene by budding and long-term expression, a quantification of GUS activity revealed that greater variability existed in budded plants compared to self-rooted mother in vitro plants for three transgenic lines. Although somatic embryogenesis provided more stable GUS activity, budding remained an efficient way of propagating transgenic plants but transgene expression in budded plants should be verified for functional analysis and further development.

Influences of aging and cloning methods on the capacity for somatic embryogenesis of a mature Hevea brasiliensis genotype.

We compared embryogenic capacities of integument explants excised from three sources of the Hevea brasiliensis (Müll. Arg.) mature genotype PB 260. The three sources were 17-year-old (BT 86) and 7-year-old (BT 96) budded trees and 7-year-old emblings (EM 96). The highest proportions of embryogenic calluses obtained from the total number of integument explants initially used were from trees of EM 96 origin, followed by BT 96 trees, with explants from BT 86 trees producing the lowest number of embryogenic calluses. Further initiation of embryogenic callus lines from the primary somatic embryos derived from the three sources was successful only for EM 96. Somatic embryo cultures from BT 86 and BT 96 sources produced only friable calluses that could not be further amplified. Overall, somatic embryo explants derived from EM 96 responded over a wider range of 3,4-dichlorophenoxyacetic acid and kinetin concentrations than the somatic embryo explants from BT 86 and BT 96 origins. The effects of chronologic, ontogenetic and physiologic aging on explant capacity for somatic embryogenesis and on the overall efficiency of the process in H. brasiliensis are discussed.

Effect of exogenous calcium on post-thaw growth recovery and subsequent plant regeneration of cryopreserved embryogenic calli of Hevea brasiliensis (Müll. Arg.).

A reliable cryopreservation technique was developed for friable embryogenic callus lines of Hevea brasiliensis. The study showed that reducing the CaCl(2) concentration of the pre-culture medium from 9 mM to 1 or 0 mM CaCl(2) before cryopreservation promoted post-thaw callus growth, 1 mM being the optimum CaCl(2) concentration for embryo regeneration. Post-thaw callus proliferation decreased in line with the increase of plated callus weight. The effect of cryopreservation was assessed on 39 independent lines showing that cryopreservation did not affect embryogenic and plant regeneration for a majority of lines. The decrease in CaCl(2) concentration of the pre-culture medium led to a drop in callus calcium content indicating a direct link between the CaCl(2) concentration of the pre-culture medium and the endogenous calcium content of the calli. It also highlighted the implication of tissue calcium content in cryotolerance. Callus water status and the different ways by which calcium could prevent cryoinjury is also discussed.