Abstract
Pathogenic strains of the soil-borne fungus Fusarium oxysporum sensu stricto infect many different plant species causing severe damage in many agricultural crops of great economic importance with serious economic and social impacts. The present study investigated the role of genes of the primary metabolism and in particular of β-amylolysis in Arabidopsis thaliana defense against the phytopathogenic fungus F. oxysporum f. sp. raphani (For). Pathogenicity tests were conducted to evaluate the susceptibility to For of A. thaliana mutants impaired to the genes encoding the four chloroplastic β-amylases BAM1, BAM2, BAM3, and BAM4, and their combinations compared to wild-type plants (wt). Among the investigated bam mutants, the genotype with disruption of BAM3, which encodes the major hydrolytic enzyme that degrades starch to maltose, was the least susceptible to For and had the smallest amount of fungal DNA in the vascular tissues using real-time qPCR. Transcriptional and metabolic analysis ...
Pathogenic strains of the soil-borne fungus Fusarium oxysporum sensu stricto infect many different plant species causing severe damage in many agricultural crops of great economic importance with serious economic and social impacts. The present study investigated the role of genes of the primary metabolism and in particular of β-amylolysis in Arabidopsis thaliana defense against the phytopathogenic fungus F. oxysporum f. sp. raphani (For). Pathogenicity tests were conducted to evaluate the susceptibility to For of A. thaliana mutants impaired to the genes encoding the four chloroplastic β-amylases BAM1, BAM2, BAM3, and BAM4, and their combinations compared to wild-type plants (wt). Among the investigated bam mutants, the genotype with disruption of BAM3, which encodes the major hydrolytic enzyme that degrades starch to maltose, was the least susceptible to For and had the smallest amount of fungal DNA in the vascular tissues using real-time qPCR. Transcriptional and metabolic analysis using microarray DNA technology and gas chromatography-mass spectrometry (GC/MS), respectively, showed that the wt and bam3 plants have different transcriptional and metabolic profiles, both in the presence and absence of the pathogen. Specifically, wt and bam3 plants inoculated with For differed in genes and metabolites related to the metabolism of carbohydrates, amino acids, plant hormones, structure and composition of cell walls. Regarding starch degradation, the expression of the following genes was studied: BAM1, BAM2, BAM3 and BAM4 encoding β-amylases, AMY3 encoding α-amylase 3 leading to the formation of linear and branched oligosaccharides, ISA3 encoding the enzyme for the hydrolysis at branching points and DPE1 encoding the D enzyme leading to glucose production or to linear oligosaccharides. BAM3 transcripts, which were significantly lower in non-inoculated (mock) bam3 plants due to the mutation, increased after inoculation with For, but they were not higher than those of wt. The above is in line with the results of the photometric measurement of starch and maltose, according to which the pathogen increased maltose and decreased starch in bam3 mutants while it decreased maltose and increased starch in wt plants. For-inoculated bam3 plants showed increased expression of BAM1, BAM2, BAM4, AMY3, ISA3 and DPE1. Therefore, inoculation with For induced the regulation of starch degradation enzymes leading to glucose production and partly to maltose production in bam3 mutants. The analysis of carbohydrate metabolism showed that For-inoculated bam3 mutants contained less D-glucose in the early stages of inoculation (3 days post inoculation), while later (7 days post inoculation) the level of the sugar was increased and was higher compared to wt plants. For inoculation increased sucrose concentration, which, through its hydrolysis to glucose, has emerged as an important molecule in plant sugar signaling networks modulating the innate immunity and defense responses during microbial attack. Transcriptional regulation of TPS6 and TPP5, genes involved in trehalose metabolism, resulted in a reduction in trehalose concentration in mutants, which is in consistent with the results of the metabolic analysis and seems to play an important role in bam3 resistance to Fusarium wilt, since trehalose and its precursor trehalose-6-phosphate (T6P) are important signaling metabolites regulating carbon assimilation and sugar status in plants stimulating plant defense response. Myo-inositol, an important sugar for many different developmental and physiological processes in eukaryotic cells, had an increased concentration in mock and For-inoculated bam3 mutants compared to wt, while the concentration of D-myo-Inositol-1-phosphate was reduced in the mutant plants after inoculation with For. MIOX1 and MIOX2 (myo-inositol oxygenase 1 and 2), that catalyze the oxidation of myo-inositol, were upregulated in inoculated bam3 plants, suggesting that For inoculation enhances the oxidative pathway of myo-inositol in mutants inducing alternative sources of glucans for maintaining metabolic homeostasis and leading to the formation of D-glucuronic acid, a precursor for the synthesis of cell wall pectic non-cellulosic compounds, especially pectin which is a key component of the primary cell wall. In confirmation of the above, the colorimetric assay for galacturonic acid (GalA) (pectin equivalent) showed that mock and For-inoculated mutants contained more pectin than the wt. The agar gel diffusion assay of protein extracts from wt and bam3 plants revealed that the pectin methylesterase (PMEs) activity was very intense in mock bam3 mutants, which is in line with the overexpression of the genes encoding pectin methylesterases PME1, PME3 and PME41 (ATPMEPCRB) in mock bam3 plants. For inoculation decreased PME activity in the early stages of inoculation (3 days post inoculation), a decrease which appears to be correlated with the underexpression of PME41 and the overexpression of PMEI7 encoding the pectin methylesterase inhibitor 7. Immunofluorescence labelling of homogalacturonan (main component of pectin) with various degrees of methylation (DM) of cell walls in stem and hypocotyls cells showed that mock bam3 plants contained more pectin, with low and high DM. In contrast, For inoculation increased the immunofluorescence of highly methylesterified pectin and decreased that of less methylesterified pectin in mutants. The above results demonstrate that the immune response of bam3 mutant against For is related to the increase of pectin content and the regulation of pectin methylation through the transcriptional regulation of genes encoding PMEs and PMEIs (PME41, PMEI7), leading to the production of highly methylesterified pectin which makes cell walls less vulnerable to For. For inoculation differentiated the expression of XTH24, XTH31 and EXP8, which encode cellulose-xyloglycan network-loosening enzymes, in bam3 plants. In particular, XTH31 was downregulated in bam3 plants at both time points post inoculation and thus it may be related to immune responses against For by preventing the fungal entry due to reduction of cell wall loosening. Concerning phytohormones, the SA signaling pathway appears to be involved in the defense of bam3 mutant against For, since the transcriptional levels of PR1 (a marker gene of SA-induced defense) were higher in mutant plants than in wt. In addition, For caused overexpression of PDF1.2, ACS8 and ERF11, genes related to ET signaling pathway, and MYC2, a transcriptional regulator of JA signaling, indicating a potential interplay between the signaling pathways of SA and ET/JA in bam3 defense against For. According to metabolic analysis, the precursors of indol-3-acetic acid (IAA), indole-3-lactic acid and 1H-indole-3-acetonitrile, increased significantly in mock and For-inoculated bam3 plants compared to the wt ones. Additionally, GH3.3, encoding a synthetase that conjugates IAA to amino acids, and DRM2, involved in auxin polar transport, were overexpressed in inoculated bam3 plants. Auxin metabolism seems to be involved in defense responses of bam3 plants against For, since GH3 proteins maintain auxin homeostasis leading to attenuation of development to prioritize defense mechanisms and DRM proteins are associated with defense responses to biotic and abiotic stress. The present study showed that BAM3 protein is a negative regulator in plant defense against F. oxysporum and can be used for genetic modification of crops in the development of Fusarium-tolerant germplasm and cultivars. The immune responses of bam3 mutants are related to the regulation of starch degradation enzymes, alterations in sugar metabolism (glucose, sucrose, trehalose and myo-inositol) and auxin metabolism, activation of an interplay between signaling pathways of SA and ET/JA and the reinforcement of cell walls in pectin with a high degree of methylation making the cell walls of bam3 mutant less vulnerable to For attack.
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