Figure 1.
Hierarchical clustering representation of genes expression quantified by qPCR in Arabidopsis roots.
Euclidean distance and average linkage were used to construct the clustering of biotic and abiotic stress responsive genes (A) and transcription factors (B). Roots were collected at 9, 24 and 48 hpi by T. asperelloides T203. Each cell represents the fold expression average of six independent biological repetitions of each time point, and is relative to control collected in each one of the time points. Black: no significant difference (P>0.05), red-up regulation, green-down regulation significantly different from control (P<0.05).
Figure 2.
Overview of modulation of expression of phyto-hormone biosynthesis and responsive genes in Arabidopsis roots during Trichoderma colonization.
In the early steps of the interaction, Trichoderma secreted MAMPs that triggered activation of signal transduction which modulated the expression of several genes. Among them, genes that have been previously shown to have a role in biosynthesis and response to JA, ethylene (Et; significant enrichment, FDR: P<0.01) and auxin (non-significant enrichment). Based on previously published data, we propose two main biological roles for the activation of those phyto-hormone related pathways in the plant-Trichoderma interaction: (i) colonization regulated by JA and ethylene, and (ii) lateral root formation regulated by JA and ethylene. Intermediate metabolites in the biosynthesis pathways are indicated by bold frame. Genes mediating the biosynthesis process are indicated without frame or marked in blue in case they show a significant (P<0.05) change in expression level upon Trichoderma colonization. Down-regulated genes are underlined. The model is based on the following published studies [28], [30], [39]–[41], [50], [76]–[78] and the oxylipins pathway (based on KEGG pathways). Gene names are in conformity with TAIR annotation.
Table 1.
Selected enriched functional categories
Figure 3.
Root colonization of different Arabidopsis genotypes by T. asperelloides.
Root colonization rate was quantified at different points as described in Materials and Methods section by qPCR (A) or fungal colonies count (CFU; B). The genotypes, WT (Col-0), wrky18/wrky40, WRKY40-HA complemented wrky18/wrky40 and fmo1 lines were assayed. Eight replicates were tested in each experiment, with 3 plants per treatments and the results are the average of three independent experiments. hpi: hours post-inoculation. *: = in each of the time points each of the line is significantly different from the other (P<0.001; t test).
Figure 4.
JAZ8 network based on co-expression analysis and data base search.
STRING version 9.0 was used to query JAZ8 (AT1G30135) gene. In the resulting network, the expression of each of the gene was monitored by qPCR 24 hpi by T. asperelloides. Red- gene that show significant increased expression (P<0.05; t test), grey- non significant change (P>0.05; t test).
Figure 5.
Expression of Arabidopsis genes after colonization by T. asperelloides.
The expression of several genes as determined by qPCR in WT (solid lines) and wrky18/wrky40 (dashed lines) plants at 9, 24 and 48 h hpi. Gene expression level was calculated with respect to control at each time point. (A) Fmo1 and CYP71A13 and PAD3 (encoding camalexin biosynthesis genes) (B) AOS and LOX2 (encoding JA-biosynthesis genes) (C) JAZ8 and JAZ10 (encoding negative regulators of JA signaling). Each time point represents the fold expression average of three independent biological repetitions, and is relative to control collected in each of the specified time points. * significant different (P<0.05; t test).
Figure 6.
qRT-PCR expression analysis of antioxidant enzymes in cucumber seedlings and roots of four weeks old Arabidopsis under salt stress.
(A) RNA was extracted from untreated Arabidopsis roots and from roots collected from plants exposed to 100 mM NaCl (NaCl), and from roots colonized by T203 for 48 hours prior exposure to 100 mM NaCl (T+NaCl). Hierarchical clustering by Euclidean distance method average linkage is shown. The color of the each cell indicates fold-change relative to control as follows: Red: significant up regulation (P<0.05; t test), green: significant down regulation (P<0.05; t test). Black: not statistically significant difference from the control,. (B) RNA was extracted from untreated cucumber seedlings (Control), from seedlings exposed to 100 mM NaCl (+NaCl), from seedlings colonized by T203 (+T) and from seedlings colonized by T203 for 48 hours prior exposure to 100 mM NaCl (+T+NaCl). RNA was extracted 1 day and 4 days after salt addition. Fold expression (for A and B) was calculated from average of three independent biological repetitions.
Table 2.
Measurement of reduced, oxidized (dehydroascorbate) and total ascorbate content in cucumber seedlings and the ASC/DHA ration
Figure 7.
Germination (%) of cucumber and Arabidopsis seedlings under salt stress conditions.
(A) Cucumber seeds were planted in untreated soil (Control) or in pots with soil mixed with a spore suspension (106spores/g soil) of T203–WT, or ACC-deaminase silenced mutants ΔACC#2 and ΔACC#3. The pots were watered with either tap water or a solution of 75 mM NaCl. Germinating seedlings were counted 7–10 days after planting. (B) Arabidopsis seeds were planted as described for the cucumber seeds and watered with either tap water, 80 mM or 125 mM NaCl solution.