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Additional file 1: Figure 1. Field design for Ganoderma infection experiments. White polybags for control plot, while red polybags for Ganoderma boninense infection. Figure 2. Performance of oil palm seedlings at 4 months after planting. Red polybags were infected Ganoderma and white polybags were control plot in the observations of growth and development of oil palm seedlings. Figure 3. PCR efficiency of Actin-101 gene. Figure 4. Melt curve analysis of the 12 targeted genes.

Hevea brasiliensis is the main source of natural rubber accounting for 42% of the worldwide rubber consumption. Latex-containing rubber particles is produced in laticifers, a specialized tissue differentiated in phloem. Latex is collected by tapping the soft bark of rubber trees. Ethephon, an ethylene releaser, is applied on bark to stimulate latex flow and regeneration between two tappings. Environmental and harvesting stresses are known to induce an oxidative stress triggering Tapping Panel Dryness (TPD). TPD is a physiological syndrome affecting latex production by promoting the agglutination of rubber particles. RNA sequencing analysis revealed an involvement of hormone signalling pathways especially ethylene and jasmonate. Given the influence of ethephon, transcriptional and post-transcriptional regulation of ethylene response factors (ERF) were characterized in response to abiotic and harvesting stresses. Some members of ERF group IX are known to be essential integrators of ethylene and jasmonate signalling pathways. The functional analysis of HbERF-IXc5 was carried out. Its overexpression in transgenic rubber trees revealed its involvement in laticifer differentiation, a biological process known to be induced by wounding and jasmonate.

Hevea brasiliensis is the main source of natural rubber accounting for 42% of the worldwide rubber consumption. Natural rubber is synthesized in rubber particles of latex cells, which are differentiated from the vascular cambia and localized in the seconda:ry phloem. Latex is collected by tapping the soft bark of rubber trees. Ethephon, an ethylene releaser, is applied on bark to stimulate latex flow and regeneration between two tappings. Above a certain threshold, environmental and harvesting stresses are known to induce an oxidative stress triggering Tapping Panel Dryness (TPD). TPD is a physiological syndrome affecting latex production through the agglutination of rubber particles. Four hundred and seven genes from 30 gene families related to reactive oxygen species (ROS) production and scavenging, and antioxidants biosynthesis were identified in the Hevea genome sequence. Based on a transcriptome analysis, 161 ROS-related genes were found expressed in latex cells. Small RNA and degradome analysis revealed 13 genes targeted by 11 microRNAs and 15 genes targeted by 16 transactingsiRNA in latex. These post-transcriptional regulations dramatically affect their gene expression profile. HbRBOH2 was identified as the main source gene of ROS in latex, while HbCuZnSOD4 might be the most important ROS scavenging enzyme for ROS detoxification in latex. Overexpression of genes encoding a superoxide dismutase (HbCuZnSOD) and an enzyme of the glutathione biosynthetic pathway (EcGSHl ) were successfully obtained in transgenic rubber plants. These later showed an increase in plant growth and their tolerance to abiotic stress. This study revealed the crucial role of antioxidant in Hevea brasiliensis laticifers and suggests seeking genetic variability for antioxidant capacity in order to improve rubber tree for the tolerance to abiotic stress and TPD occurrence.

Reverse Transcriptase Quantitative PCR (RTqPCR) is a powerful technique in order to detect low abundance of mRNA in the plant cell. The measurement of transcript abundance is relative to the control of expression such as housekeeping genes. Therefore, the reliability of RT-qPCR depends essentially to the choice of these internal controls also called reference genes. That is the reason why a prior validation of reference genes is suggested for every set of cDNA samples used in a new RT-qPCR experiment. This study aimed to analyze the stability of eleven selected housekeeping genes in three Hevea brasiliensis tissues (leaf, bark and root) under15 days of moderate water deficit. Total RNA was isolated from 18 samples consisting of control and stressed-plants collected at day-0 (D0), day-5 (D5) and day-15 (D15).The quality of cDNA synthesized was examined by PCR using HbActin primer. The eleventh primers encoding Hevea housekeeping genes (HbActin, HbelF1Aa, HbUBC4, HbUBC2b, HbYLS8, HbRH2b, HbRH8, HbUBC2a, HbαTub, Hb40S and HbUBI) were validated using PCR amplification. The Crossing-point (Cp) values were measured using a second derivative method after RT-qPCR analysis revealing a significantly higher Cp mean values for 11 housekeeping genes at D5 compared to D0 and D15 sampling points. This study suggests that a simple coefficient of variation (CV) method can be used to rank Hevea reference genes based on its stable expression. Five housekeeping genes (HbRH2b, HbRH8, HbUBC4, HbαTUB and HbActin) can be used for RT-qPCR analysis in Hevea brasiliensis under moderate water deficit. The HbRH2b gene was the most stable among others.

Real-Time quantitative RT-PCR technique is a sensitive method for measuring the accumulation of gene transcripts. This widely used technique in a variety of plant species; including rubber tree (Hevea brasiliensis) must meet basic criteria in order to produce accurate gene expression markers. Gene expression markers associated to the response of ethephon stimulation such as the Hevea brasiliensis Ethylene Response Factors (HbERFs) family has been characterized in a single rubber clone. It is known that the effect of genotype on rubber tree clones can give different expression of the same gene. This difference can be converted into a profile that characterizes clones to a certain trait. This study aimed to identify gene expression profile in response to ethephon stimulation using six HbERFs (HbORA47, HbRAP2.3, HbERF12, HbERF3, HbABR1, HbRRTF1) in three rubber tree clones having contrasted latex metabolism (PB 260, SP 217, and RRIM 600). Total RNA was isolated from 18 samples and used for cDNA synthesis. The quality of cDNAs was examined by PCR using HbActin primer. HbRH2b was selected among the 11 housekeeping genes to be used as an internal control in gene expression analysis. Gene expression analysis resulted to an induction and inhibition of HbERFs by ethephon stimulation which are specific to a particular clone. Expression profile of three Hevea clones showed distinct characteristics. The high latex metabolism clone PB 260 was characterized by the upregulated expression of HbRAP2.3 and HbERF12. The low latex metabolism clone SP 217 was characterized by the upregulated expression of HbRAP2.3 and HbRRTF1. Meanwhile, the profile of intermediate latex metabolism clone RRIM 600 was shown by downregulated expression of HbORA47 and up-regulated expression of HbABR1. This study shows that HbERFs gene family is an important expression marker because it can inform physiological conditions of rubber clones associated in response to ethephon.

Les racines jouent un rôle dans la stabilité de la plante, mais aussi dans l'absorption et le transport de l'eau et des éléments minéraux. Lors d'un stress hydrique, elles sont affectées et servent comme organes de perception de signaux. Pourtant, le développement d'un système racinaire est spécifique chez les espèces dicotylédones pérennes, par rapport à celles de modèle connues. Certaines études ont été réalisées chez quelques espèces pérennes au niveau moléculaire afin d'identifier les gènes potentiels impliqués dans le stress hydrique. Cependant, les véritables connaissances sur les différents types de racines dans un système racinaire sont toujours manquantes. L'objectif de cette étude est donc de créer une cartographie spatiale de l'expression génique de collection des gènes disponibles dans les différents types de racines chez une espèce dicotylédone pérenne (Hevea brasiliensis) en réponse à un déficit hydrique par une approche de RT-PCR en temps réel. Premièrement, en s'appuyant de la base de données d'EST des racines d'hévéa contenant 50146 contigs, sept gènes ayant une forte homologie avec des gènes impliqués dans le développement racinaire ont pu être identifiés. Ces gènes ont été ajoutés à une collection de gènes cibles chez l'hévéa. Deuxièmement, sur la base des 48 gènes cibles de cette collection, l'ensemble de notre résultat a montré que la régulation spatiale de ces gènes est exacerbée par le déficit hydrique modéré (FTSW=0,4) (13 gènes chez les plantes témoins contre 22 chez les plantes traitées) dans de différents organes étudiés. Les niveaux d'abondance relative des transcrits en réponse au déficit hydrique varient significativement au sein d'un même organe pour moins de gènes (10 gènes). Au niveau des organes racinaires, les racines extra-fines sont révélées comme organe très sensible au déficit hydrique.

L'hévéa est l'arbre le plus haut producteur de caoutchouc naturel qui est un hydrocarbure de type polyisoprène à haut poids moléculaire présent dans le latex. Dans la plantation, la production de latex par la saignée est stimulée par l'application d'éthéphon. Cet éthylène exogène provoque la biosynthèse d'éthylène endogène. La saignée, la stimulation ainsi que les stress environnementaux déclenche la production de radicaux libres. Ainsi, l'activation des systèmes de détoxication des espèces activées de l'oxygène est importante pour la stabilité de la production de latex. La régulation de l'expression de gènes intervenant dans la biosynthèse de l'éthylène et les systèmes antioxydants ont été étudiées dans les racines en réponse à la blessure, l'éthylène exogène et le stress hydrique afin d'avoir une meilleure compréhension des processus développementaux et des réponses aux stress. En effet, aucune information n'est disponible sur le fonctionnement moléculaire des racines chez l'hévéa. Les gènes intervenant dans la biosynthèse de l'éthylène et les systèmes de détoxication des espèces activées de l'oxygène ont leur expression régulée différentiellement sur le plan spatio-temporel et en réponse aux stress abiotiques. Il y a une grande diversité d'accumulation de transcrits entre les trois organes étudiés (feuille, écorce et racine). Par exemple, les transcrits SAMS sont fortement accumulés dans l'écorce, ceux d'ACO3 dans les feuilles. Pour les systèmes antioxydants, les transcrits CAS sont accumulés dans les écorces et les racines, ceux de MnSOD dans les feuilles, d'APX1 dans tous les organes, et de GCLchI dans les racines.