Transgenic plants that express the phytoplasma effector SAP11 show altered phosphate starvation and defense responses

Abstract

Phytoplasmas have the smallest genome among bacteria and lack many essential genes required for biosynthetic and metabolic functions, making them unculturable, phloem-limited plant pathogens. In this study, we observed that transgenic Arabidopsis (Arabidopsis thaliana) expressing the secreted Aster Yellows phytoplasma strain Witches' Broom protein11 shows an altered root architecture, similarly to the disease symptoms of phytoplasma-infected plants, by forming hairy roots. This morphological change is paralleled by an accumulation of cellular phosphate (Pi) and an increase in the expression levels of Pi starvation-induced genes and microRNAs. In addition to the Pi starvation responses, we found that secreted Aster Yellows phytoplasma strain Witches' Broom protein11 suppresses salicylic acid-mediated defense responses and enhances the growth of a bacterial pathogen. These results contribute to an improved understanding of the role of phytoplasma effector SAP11 and provide new insights for understanding the molecular basis of plant-pathogen interactions.

Figure 1.

Expression of SAP11_AYWB regulates the accumulation of miRNAs involved in Pi- and auxin-signaling responses in Arabidopsis. A, Comparison of leaf morphologies between wild-type (Col-0) and transgenic plants carrying SAP11_AYWB or miR319a driven by Cauliflower mosaic virus 35S promoter. Bar = 8 mm. B, Examination of the translated product of SAP11_AYWB by western blotting using specific antibody against SAP11_AYWB. Asterisk indicates the cross-reacting band appeared in all samples. Antitubulin was used for loading control. C, Comparisons of the expression levels of miRNAs in the wild type and 35S::SAP11_AYWB transgenic lines by small RNA northern blotting. The RNAs staining bands were used as a loading control. The values of band intensities were measured using ImageJ.

Figure 2.

SAP11_AYWB elicits the expression of Pi starvation-induced genes and increases the accumulation of Pi in Arabidopsis. A, The mRNA levels of genes triggered by Pi starvation responses in wild-type (Col-0) plants and 35S::SAP11_AYWB transgenic lines were examined by qRT-PCR and normalized to Actin2. The relative expression levels of each gene in wild-type plants were set at 1. B, The levels of Pi concentration in the aerial parts of the wild type and 35S::SAP11_AYWB transgenic lines grown in hydroponic solutions with 0.01 or 0.25 mm KH₂PO₄ were measured. Statistically significant differences were determined using Student’s t test (*P < 0.05, **P < 0.005 for transgenic plants versus the wild type).

Figure 3.

Expression of SAP11_AYWB in Arabidopsis alters the phenotypes in anthocyanin accumulation and root architecture. Comparisons of plant size (A), anthocyanin accumulation (B), and root morphology (C) between wild-type (Col-0) plants and 35S::SAP11_AYWB transgenic lines grown on horizontal (A) or vertical (C) plates containing 0.01 or 0.25 mm KH₂PO₄. Statistically significant differences were determined using Student’s t test (*P < 0.05, **P < 0.005 for transgenic plants versus the wild type). FW, Fresh weight. Bars = 15 mm.

Figure 4.

Comparison of gene expression profiles. A, Comparison of the expression levels of SAP11_AYWB in Arabidopsis 35S::SAP11_AYWB transgenic lines by western blotting using specific antibodies against SAP11_AYWB. Asterisk indicates the cross-reacting band appeared in all samples. B, Venn diagrams show the comparisons of SAP11_AYWB-elicited genes (P < 0.001) with the Pi starvation response (PSR) genes (up-regulation, 2-fold higher). Pi starvation response (I) indicates the data set collected by Müller et al. (2007) from leaf samples in response to Pi starvation. Pi starvation responses (II) and (III) indicate the data set collected by Liu et al. (2011) from leaf and root samples, respectively, in response to Pi starvation. C, The mRNA levels of differentially expressed genes identified in the 35S::SAP11_AYWB transgenic line number 13 were examined by qRT-PCR and normalized to Actin2. The relative expression levels of each gene in wild-type (Col-0) plants were set at 1. D, Comparisons of the expression levels of miRNAs by small RNA northern blotting. The RNAs staining bands were used as a loading control. E, The mRNA levels of genes involved in Pi starvation responses were examined by qRT-PCR and normalized to Actin2. The relative expression levels of each gene in wild-type (Col-0) plants were set at 1. Statistically significant differences were determined using Student’s t test (*P < 0.05, **P < 0.005 for transgenic plants versus the wild type). The values of band intensities were measured using ImageJ.

Figure 5.

Comparison of miRNA expression profiles. Scatter plot shows the related expression levels of known miRNAs in Arabidopsis wild-type (WT) plants (x axis) and 35S::SAP11_AYWB transgenic line number 13 (y axis), in which the scales indicate the expression intensity of normalized miRNAs. Red points refer to miRNAs whose expression levels were 2-fold higher in 35S::SAP11_AYWB transgenic line number 13 as compared with wild-type plants. Green points refer to miRNAs whose expression levels were 2-fold lower in 35S::SAP11_AYWB transgenic line number 13 as compared with wild-type plants. Blue points refer to miRNAs whose expression levels were not higher or lower than 2-fold in the 35S::SAP11_AYWB transgenic line number 13 as compared with wild-type plants.

Figure 6.

Phenotypic comparisons in leaf morphology and root architecture between 35S::miR319a and 35S::SAP11_AYWB transgenic plants. A, Leaf morphologies of Arabidopsis seedlings grown on 1/2× MS medium. Bar = 8 mm. B, Root architectures of Arabidopsis seedlings grown on the vertical plate containing 0.01 mm KH₂PO₄. Bar = 15 mm.

Figure 7.

PHR1 is required for SAP11_AYWB-triggered Pi starvation responses in Arabidopsis. A, Leaf morphologies of Arabidopsis transgenic plants overexpressing SAP11_AYWB in the background of a phr1 mutant. The expression level of SAP11_AYWB was detected by western blotting using specific antibodies against SAP11_AYWB. Asterisk indicates the cross-reacting band appeared in all samples. Bar = 8 mm. B, The mRNA levels of genes involved in Pi starvation responses among wild-type (Col-0), phr1 mutant, and 35S::SAP11_AYWB/phr1 transgenic plants were examined by qRT-PCR and normalized to Actin2. The relative expression levels of each gene in wild-type plants were set at 1. Statistically significant differences were determined using Student’s t test (*P < 0.05, **P < 0.005 for the wild type versus phr1; ⁺P > 0.05 for phr1 versus 35S::SAP11_AYWB/phr1).

Figure 8.

SAP11_AYWB suppresses the innate immunity against bacterial pathogens in Arabidopsis. RNA samples were extracted from SA-treated wild-type (Col-0) and 35S::SAP11_AYWB transgenic plants harvested at indicated times. The mRNA levels of genes involved in SA-mediated defense signaling network (A) and JA-signaling responses (B) were examined by qRT-PCR and normalized to Actin2. The relative expression levels of each gene in wild-type plants without SA treatment were set at 1. C, Bacterial growth of Pto DC3000 in wild-type and 35S::SAP11_AYWB transgenic plants were measured to examine the effects of SAP11_AYWB on the resistance of Arabidopsis against bacterial pathogens. Hand-infiltrated leaves were collected at the indicated times for measuring the in planta growth of Pto DC3000. Statistically significant differences were determined using Student’s t test (*P < 0.05, **P < 0.005 for transgenic plants versus the wild type).