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. 2019 Sep 10;20(1):703.
doi: 10.1186/s12864-019-6062-x.

'Candidatus Phytoplasma solani' interferes with the distribution and uptake of iron in tomato

Sara Buoso  1 , Laura Pagliari  1 , Rita Musetti  1 , Marta Martini  1 , Fabio Marroni  1   2 , Wolfgang Schmidt  3   4 , Simonetta Santi  5
Affiliations

'Candidatus Phytoplasma solani' interferes with the distribution and uptake of iron in tomato

Sara Buoso et al. BMC Genomics. .

Abstract

Background: 'Candidatus Phytoplasma solani' is endemic in Europe and infects a wide range of weeds and cultivated plants. Phytoplasmas are prokaryotic plant pathogens that colonize the sieve elements of their host plant, causing severe alterations in phloem function and impairment of assimilate translocation. Typical symptoms of infected plants include yellowing of leaves or shoots, leaf curling, and general stunting, but the molecular mechanisms underlying most of the reported changes remain largely enigmatic. To infer a possible involvement of Fe in the host-phytoplasma interaction, we investigated the effects of 'Candidatus Phytoplasma solani' infection on tomato plants (Solanum lycopersicum cv. Micro-Tom) grown under different Fe regimes.

Results: Both phytoplasma infection and Fe starvation led to the development of chlorotic leaves and altered thylakoid organization. In infected plants, Fe accumulated in phloem tissue, altering the local distribution of Fe. In infected plants, Fe starvation had additive effects on chlorophyll content and leaf chlorosis, suggesting that the two conditions affected the phenotypic readout via separate routes. To gain insights into the transcriptional response to phytoplasma infection, or Fe deficiency, transcriptome profiling was performed on midrib-enriched leaves. RNA-seq analysis revealed that both stress conditions altered the expression of a large (> 800) subset of common genes involved in photosynthetic light reactions, porphyrin / chlorophyll metabolism, and in flowering control. In Fe-deficient plants, phytoplasma infection perturbed the Fe deficiency response in roots, possibly by interference with the synthesis or transport of a promotive signal transmitted from the leaves to the roots.

Conclusions: 'Candidatus Phytoplasma solani' infection changes the Fe distribution in tomato leaves, affects the photosynthetic machinery and perturbs the orchestration of root-mediated transport processes by compromising shoot-to-root communication.

Keywords: Carotenoids metabolism; Chlorophyll; Iron deficiency; Leaves; NGS; Phloem; Phytoplasma; Porphyrin; Roots; Tomato.

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Conflict of interest statement

The authors declare that they have no competing interets.

Figures

Fig. 1
Fig. 1
Phenotypes of representative tomato plants grown under different experimental conditions. a-d Whole plants, leaves, shoots, and roots of (a) healthy Fe-sufficient (H/+Fe) plants, b infected Fe-sufficient (I/+Fe) plants (c) healthy Fe-deficient (H/−Fe) plants, and (d) infected Fe-deficient plants, (I/−Fe). e Total plant fresh weight. Results are expressed as mean ± SD (n = 6). f Leaf SPAD index values of fully expanded leaves. Results are expressed as mean ± SD (n = 150). g Leaf surface area. Results are expressed as mean ± SD (n = 30). Different letters indicate statistically significant differences (P < 0.05) among conditions (one-way ANOVA followed by Holm-Sidak’s test)
Fig. 2
Fig. 2
Relative quantification of ‘Ca. P. solani’ in infected Fe-sufficient and Fe-deficient tomato leaves. The amount of ‘Ca. P. solani’ DNA was determined by qPCR analysis of the 16SrRNA gene of ‘Ca. P. solani’ relative to the tomato single-copy gene Chloronerva. Results are expressed as mean ± SD (n = 8). Different letters indicate statistically significant differences (P < 0.05) among conditions (one-way ANOVA followed by Holm-Sidak’s test)
Fig. 3
Fig. 3
Effects of Fe starvation and phytoplasma infection on phloem ultrastructure. a-d Micrographs of healthy (H/+Fe) plants (a, b) and infected (I/+Fe) plants (c, d); phytoplasmas were detected exclusively in the lumen of the sieve elements (c). In companion and mesophyll cells, chloroplasts showed distorted arrangement of thylakoid stacks and significative accumulation of starch (d). e-g Micrographs of healthy Fe-starved (H/−Fe) tissues (e, f); sieve elements exhibited a regular ultrastructure (e), companion and parenchyma cells exhibited misshaped chloroplasts with large starch grains embedded between granal and stromal lamellae (f). g, h Phytoplasma-infected/Fe-starved (I/−Fe) plants with phytoplasma in sieve elements (g) and severely altered chloroplast ultrastructure (h). cc: companion cell; ch: chloroplast; i: inset; pc: parenchyma cell; se: sieve element; *: starch; arrowheads indicate phytoplasmas. Three non-serial cross sections from five plants were analysed for each condition (n = 15)
Fig. 4
Fig. 4
Effects of phytoplasma infection and Fe starvation on Fe concentration in whole leaves, midribs and roots. Fe concentration in whole leaves, leaf midribs and roots of H/+Fe, I/+Fe, H/−Fe, and I/−Fe tomato plants. Fe concentration was determined by ICP-OES. Results are expressed as mean ± SD (n = 6). DW: dry weight. Different letters indicate statistically significant differences (P < 0.05) among conditions (one-way ANOVA followed by Holm-Sidak’s test)
Fig. 5
Fig. 5
Effects of phytoplasma infection and Fe starvation on Fe distribution in the leaf midrib and surrounding parenchyma. a-d Perls’-DAB staining on 7 μm-thick transversal sections of leaf tissues in the phloem area of healthy (H/+Fe) plants (a), phytoplasma-infected (I/+Fe) plants (b), Fe-starved (H/−Fe) plants (c), and phytoplasma-infected/Fe-starved (I/−Fe) plants (d). e-h Longitudinal sections. Fe dots (arrow heads) are visualized in phloem cells of healthy and phytoplasma-infected/Fe-sufficient plants. i-n transversal sections of the xylem area. Fe dots are visualised only in H/+Fe plants. ph: phloem; x: xylem; arrowheads indicate Fe dots. Scale bars: 10 μm
Fig. 6
Fig. 6
Venn diagram, KEGG pathway, and gene ontology (GO) enrichment analyses of differentially expressed genes (DEGs) in the three comparison groups. a Venn diagram showing the number of up-, down-, and anti-directionally regulated differentially expressed genes (DEGs) that were common and specific for the pairwise comparisons. b KEGG pathway enrichment. X-axis indicates the value of -Log10 (q value). c GO enrichment. The y-axis indicates the percentage of significant DEGs corresponding to the total number of genes annotated in each GO category (P < 0.05). DEGs were grouped into three major functional categories: biological process, cellular component, and molecular function
Fig. 7
Fig. 7
Heat map analysis of differentially expressed genes. a-d Fold changes of DEGs in the three comparison groups involved in antenna protein cluster (KEGG sly00196; a), photosynthesis-light reactions (sly00195; b), porphyrin and chlorophyll metabolism (sly00860; c), and carotenoid biosynthesis (sly00906; d). e, f Partial representation of porphyrin and chlorophyll (e) and carotenoid biosynthesis (f) pathways
Fig. 8
Fig. 8
Expression analysis of Fe-related genes in tomato roots by RT-qPCR. The mean normalized expression (MNE) of each gene is plotted as the transcript abundance compared with the UPL3 expression level (set at 100). Results are expressed as mean ± SD (n = 5). Different letters indicate statistically significant differences (P < 0.05) among the conditions (one-way ANOVA followed by Holm-Sidak’s test)
Fig. 9
Fig. 9
Model summarizing the effect of phytoplasma infection and/or Fe deficiency on Fe content and Fe acquisition. In healthy tomato plants grown on Fe-sufficient conditions (H/+Fe), Fe is distributed in the whole leaf. In Fe-sufficient, infected leaf (I/+Fe), Fe content is increased in the midrib, concentrating in the phloem tissue. This shift towards the infection site does not induce any root response. Independent of their health status, Fe-deficient plants H/−Fe and I/−Fe) show an extremely reduced Fe content, with no peculiar distribution. In these conditions, the Fe acquisition mechanism is induced, but less induced when the phloem is infected. An impairment of the phloem mass flow and/or an interference with signals moving in the phloem are suggested for phytoplasma infection
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