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Klim Dyachkov
Klim Dyachkov

My Sweet Orange Tree.pdf

After Zeze's father gets fired, the whole family moves, and the new house has a few trees that each of the siblings claims. After having all the trees taken, Zeze gets upset, but one of his older sisters, Gloria, suggests looking in the backyard and Zeze soon discovers a small orange tree. At first, Zeze doesn't like his tree, but he finds out that amazingly, he can communicate with it. He gives the tree a name, Pinkie, and the two become best friends.

My Sweet Orange Tree.pdf

With financial difficulties, the family has to move. At the new home, Zezé finds an orange tree, which he talks every day. However, for being extremely extrovert he got involved in several confusions. One of them, he tries to ride on the bumper of Manoel "Portuga" Valadares, but is caught and spanked. The boy feels humiliated and wants revenge, however Valadares ends up understanding Zezé, which turns to share his world of fantasies, and a new friendship arises.[6]

Zezé also has a broad imagination to match his resourcefulness. When the family moves house, Zezé claims a sweet-orange tree in the garden for himself. He names it Pinkie, imagines he can hear it talk, and whiles away hours riding in its branches with Tom Mix and other movie cowboys of the day.

In the present study, a whole-genome transcriptional analysis of rough lemon and sweet orange leaves was conducted at 7 weeks post-inoculation in order to identify genes that were induced as part of an early response to CLas. Leaf samples collected immediately after inoculation (week 0) were used as a baseline, which was not done in previous transcriptome studies of CLas in citrus.4,10,11 By using week 0 as a baseline, the natural variation among biological replications could be minimized and the number of genes whose expression was significantly affected by CLas could be maximized. The response of mock-inoculated and CLas-inoculated rough lemon and sweet orange were separately analyzed and the two datasets were subsequently compared, in order to identify biological mechanisms associated with each genotype and infection versus mock-inoculation. The transcriptome analysis identified statistically significant differentially expressed genes (DEGs) between rough lemon and sweet orange in response to CLas. A distinct difference in the defense response between rough lemon and sweet orange in CLas-inoculated leaves was observed. Not only were two distinct sets of DEGs identified but greater amplitude in the profile of defense response genes in rough lemon, relative to sweet orange, was also observed. The present study provides a comprehensive overview of the early transcriptional reprogramming that occurs in rough lemon in response to CLas.

Leaf samples collected at week 7 did not exhibit the characteristic blotchy mottle appearance and were RT-qPCR negative for CLas. Continued RT-qPCR analysis for CLas was conducted every two weeks after inoculation. Positive confirmation of CLas in inoculated plants was not obtained in rough lemon and sweet orange until 23 weeks post inoculation. Even after 8 months, however, rough lemon did not exhibit any signs of growth inhibition, and continued growth of new shoots with few or no symptoms was observed; however, the typical blotchy mottled appearance was commonly found on mature, older leaves (Figure 1). In contrast, severe levels of blotchiness were observed on mature and older leaves of CLas- inoculated sweet orange and growth was significantly inhibited, with the rare production of new shoots toward the end of the experiment (Figure 1). CLas was not detected by RT-qPCR in samples from any of the mock-inoculated rough lemon or sweet orange throughout the entire experiment.

Venn diagrams of differentially expressed genes in mock- and CLas-inoculated rough lemon (a) and sweet orange (b). The number of significantly up-regulated genes is shown (first) in red, and significantly downregulated genes (second) in blue.

Distribution of significant differentially expressed genes (DEGs): (a) CLas-inoculated rough lemon; (b) Mock-inoculated rough lemon; (c) CLas-inoculated sweet orange; (d) Mock-inoculated sweet orange.

An appropriate statistical test is needed to determine whether or not observed differences in the number of obtained sequences (read counts) of a specific gene is significant, that is, whether the difference is greater than what would be expected due to natural random variation.22 In addition, it is also critical to design the experiment so that natural variation (not due to a treatment effect) in gene expression is minimized and the number of identified DEGs is maximized. Previous studies directly comparing CLas- and mock-inoculated plants, or tolerant and susceptible genotypes, may have not sufficiently reduced biological variation after inoculation at week 0. As a result, a low number of early-stage DEGs were identified.4,10,11,27 In the present study, gene expression in week 0 was used as a baseline in order to reduce the level of random variation between individual plants. To identify the DEGs induced by CLas, only DEGs that were uniquely expressed in CLas-inoculated and not in mock-inoculated samples at week 7 were selected for comparative analysis. Using this approach, we identified a greater number of DEGs in response to CLas inoculation than by direct pairwise comparisons of CLas-inoculated rough lemon and sweet orange at week 7. This approach has not been used in previous transcriptome studies of CLas infected citrus. However, we would not identify as DEGs those genes which had high absolute levels of expression in both week 0 and week 7 after inoculation. We speculate that some of these genes might play important roles against CLas, although these genes were not necessarily induced by CLas.

The peptide flg22, a bacterial flagella protein recognized by most plants, activates a MAPK cascade which then leads the activation of WRKY-type transcription factors, key regulators of plant defense.41 Flagella in CLas, however, have not been observed in any of the numerous electron micrographs of these bacteria infecting plants and psyllids.2 The reduced genome of CLas and their transmission by psyllids may allow it to avoid PTI. CLas, however, still possesses 57 genes coding for products that function in cell envelope biogenesis and the outer membrane, including lipopolysaccharides, and most flagellar genes, which might function as PAMPs.33 Elongation factor Tu (EF-Tu) is one of the most abundant bacterial proteins and is recognized as a PAMP by Arabidopsis.42 The plant PRR for EF-Tu is the LRR-RLK EF-Tu receptor (EFR), which belongs to the same subfamily (LRRXII) as FLS2.42 In this study, upregulation (LFC=2.84) of EFR was only found in inoculated sweet orange. No expression of FLS2 was observed in CLas-inoculated leaves of either rough lemon or sweet orange.

During PTI, activation of the MAPK cascade leads to the activation of WRKY-type transcription factors and other key regulators of plant immunity.43 It is thought that the MAPK cascade regulates plant immunity through the activation of defense-related genes via direct phosphorylation of downstream transcription factors, such as WRKYs and ERFs.44,45 MKK9 is upstream of MPK3, and MAPKKK19 is upstream of MKK9 (Figure 5). In our study rough lemon exhibited a stronger MAPK response than sweet orange (Table 1). After the perception of flg22, MPK6 activates ethylene biosynthesis through the phosphorylation of ACS6.46 ACS6 was highly upregulated in CLas-inoculated rough lemon but not sweet orange. We also observed the strong upregulation of ERF1 (LFC=3.18) and ERF9 (LFC=3.31) in CLas-inoculated leaves of rough lemon (Tables 3a and b, Figure 5). In response to pathogen attack, ET and JA cooperate through transcriptional induction of ET response factor 1 (ERF1).47 Activation of the MAPK cascade induces members of the WRKY family of transcription factors and defense-related genes 48 in tobacco.49 For example, WRKY22 is activated via a MAPKcascade induced by flg22.50 In our study, WRKY22 expression was only found in sweet orange, however, the upregulation was only moderate (LFC=1.96). These data were much lower than the upregulation of group 1 WRKY (WRKY33, WRKY70 and WRKY40) TFs in rough lemon. Emerging evidence has indicated that group I WRKY transcription factors, which contain a conserved motif in the N-terminal region, are also activated by MAPK-dependent phosphorylation, underlining their importance in plant immunity.51

MAPK cascades (Figure 5) have also been shown to be involved in ABA signaling.55 In the present study, MAK 3, MAPKKK19 and MKK9 were all significantly upregulated in CLas-inoculated rough lemon (Supplementary Files, Supplementary Figures S1 and S2), and may play a role in the ABA signaling pathway as suggested in a previous Arabidopsis study. Blast2GO also indicated strong over representation of an ABA signaling GO term in CLas-inoculated rough lemon (Supplementary File 6). MPK3 is activated by both H2O2 and ABA in Arabidopsis seedlings, and overexpression of MPK3 increased ABA sensitivity to ABA-induced post-germination arrest of growth.56 MPK9, which is preferentially expressed in guard cells, is also activated by ABA and has been shown to mediate ABA signaling in guard cells.57 MPK3/MPK6 negatively regulates ABA signaling in plants.58 The WRKY superfamily of TFs is the major regulator of plant defense and SA-mediated signaling, but also participates in ABA-mediated signaling.59 AtWRKY40 has been reported to be a negative regulator of ABA signaling during seed germination and also interacts with AtWRKY18 and AtWRKY60 to inhibit the expression of stress-responsive genes.53 In our study, WRKY40 was highly upregulated (LFC=4.9) in CLas-inoculated rough lemon. In addition, it was the most highly expressed among all of the upregulated WRKYs in CLas-inoculated rough lemon, but was not expressed in inoculated sweet orange. An and Mou32 stated that although ABA is known to play a crucial role in adaptation to abiotic stress, its role in biotic stress responses is less understood. In general, however, ABA is considered to be a negative regulator of disease resistance.55


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