Development of a Mild Viral Expression System for Gain-Of-Function Study of Phytoplasma Effector In Planta

PHYL1 and SAP54 are orthologs of pathogenic effectors of Aster yellow witches’-broom (AYWB) phytoplasma and Peanut witches’-broom (PnWB) phytoplasma, respectively. These effectors cause virescence and phyllody symptoms (hereafter leafy flower) in phytoplasma-infected plants. T₀ lines of transgenic Arabidopsis expressing the PHYL1 or SAP54 genes (PHYL1 or SAP54 plants) show a leafy flower phenotype and result in seedless, suggesting that PHYL1 and SAP54 interfere with reproduction stage that restrict gain-of-function studies in the next generation of transgenic plants. Turnip mosaic virus (TuMV) mild strain (TuGK) has an Arg182Lys mutation in the helper-component proteinase (HC-Pro^R182K) that blocks suppression of the miRNA pathway and prevents symptom development in TuGK-infected plants. We exploited TuGK as a viral vector for gain-of-function studies of PHYL1 and SAP54 in Arabidopsis plants. TuGK-PHYL1- and TuGK-SAP54-infected Arabidopsis plants produced identical leafy flower phenotypes and similar gene expression profiles as PHYL1 and SAP54 plants. In addition, the leafy flower formation rate was enhanced in TuGK-PHYL1- or TuGK-SAP54-infected Arabidopsis plants that compared with the T₀ lines of PHYL1 plants. These results provide more evidence and novel directions for further studying the mechanism of PHYL1/SAP54-mediated leafy flower development. In addition, the TuGK vector is a good alternative in transgenic plant approaches for rapid gene expression in gain-of-function studies.     

Defining subdomains of the K domain important for protein–protein interactions of plant MADS proteins

The MADS proteins APETALA3 (AP3), PISTILLATA (PI), SEPALLATAI (SEPI), SEP2, SEP3, AGAMOUS, and APETALA are required for proper floral organ identity in Arabidopsis flowers. All of these floral MADS proteins conserve two domains: the MADS domain that mediates DNA binding and dimerization, and the K domain that mediates protein protein interaction. The K domain is postulated to form a several amphipathic c-helices referred to as K1, K2, and K3. The K1 and K2 helicies are located entirely within the K domain while the K3 helix spans the K domain-C domain boundary. Here we report on our studies on the interactions of the B class MADS proteins AP3 and PI with the E class MADS proteins SEP1, SEP2, and SEP3. A comparative analysis of mutants in the K domain reveals that the subdomains mediating the PI/AP3 interaction are different from the subdomains mediating the PI/SEP3 (or PI/SEP1) interaction. The strong PI/SEP3 (or PI/SEP1) interaction requires K2, part of K3, and the interhelical region between K1 and K2. By contrast, K1, K2 and the region between K1 and K2 are important for strong AP3/PI interaction. Most of the K3 helix does not appear to be important for either the PI/AP3 or the PI/SEP3 (or PI/SEP1) interaction. Conserved hydrophobic positions are most important for the strength of both PI/AP3 and PI/SEP3 dimerization, though ionic and/or polar interactions appear to play a secondary role.     

Phytoplasma effector SAP54 induces indeterminate leaf-like flower development in Arabidopsis plants

Phytoplasmas are insect-transmitted bacterial plant pathogens that cause considerable damage to a diverse range of agricultural crops globally. Symptoms induced in infected plants suggest that these phytopathogens may modulate developmental processes within the plant host. We report herein that Aster Yellows phytoplasma strain Witches' Broom (AY-WB) readily infects the model plant Arabidopsis (Arabidopsis thaliana) ecotype Columbia, inducing symptoms that are characteristic of phytoplasma infection, such as the production of green leaf-like flowers (virescence and phyllody) and increased formation of stems and branches (witches' broom). We found that the majority of genes encoding secreted AY-WB proteins (SAPs), which are candidate effector proteins, are expressed in Arabidopsis and the AY-WB insect vector Macrosteles quadrilineatus (Hemiptera; Cicadellidae). To identify which of these effector proteins induce symptoms of phyllody and virescence, we individually expressed the effector genes in Arabidopsis. From this screen, we have identified a novel AY-WB effector protein, SAP54, that alters floral development, resulting in the production of leaf-like flowers that are similar to those produced by plants infected with this phytoplasma. This study offers novel insight into the effector profile of an insect-transmitted plant pathogen and reports to our knowledge the first example of a microbial pathogen effector protein that targets flower development in a host.     

Unique morphological changes in plant pathogenic phytoplasma-infected petunia flowers are related to transcriptional regulation of floral homeotic genes in an organ-specific manner

Abnormal flowers are often induced by infection of certain plant pathogens, e.g. phytoplasma, but the molecular mechanisms underlying these malformations have remained poorly understood. Here, we show that infection with OY-W phytoplasma (Candidatus Phytoplasma asteris, onion yellows phytoplasma strain, line OY-W) affects the expression of the floral homeotic genes of petunia plants in an organ-specific manner. Upon infection with OY-W phytoplasma, floral morphological changes, including conversion to leaf-like structures, were observed in sepals, petals and pistils, but not in stamens. As the expression levels of homeotic genes differ greatly between floral organs, we examined the expression levels of homeotic genes in each floral organ infected by OY-W phytoplasma, compared with healthy plants. The expression levels of several homeotic genes required for organ development, such as PFG, PhGLO1 and FBP7, were significantly downregulated by the phytoplasma infection in floral organs, except the stamens, suggesting that the unique morphological changes caused by the phytoplasma infection might result from the significant decrease in expression of some crucial homeotic genes. Moreover, the expression levels of TER, ALF and DOT genes, which are known to participate in floral meristem identity, were significantly downregulated in the phytoplasma-infected petunia meristems, implying that phytoplasma would affect an upstream signaling pathway of floral meristem identity. Our results suggest that phytoplasma infection may have complex effects on floral development, resulting in the unique phenotypes that were clearly distinct from the mutant flower phenotypes produced by the knock-out or the overexpression of certain homeotic genes.     

pistillata-5, an Arabidopsis B class mutant with strong defects in petal but not in stamen development

The Arabidopsis floral organ identity genes APETALA3 (AP3) and PISTILLATA (PI) encode related DNA-binding proteins of the MADS family. Considerable evidence supports the hypothesis that a heterodimer of AP3 and PI is an essential component of B class activity. All ap3 and pi alleles characterized to date exhibit equivalent phenotypic defects in both whorls 2 and 3. In strong ap3 and pi mutants, petals and stamens are missing and sepals and carpels develop in their place. Weak ap3 and pi mutants exhibit partial conversions of petals to sepals and stamens to carpels. In this report, we describe the isolation and characterization of pi-5, an unusual B class mutant that exhibits defects in whorl 2 where sepals develop in place of petals, but third whorl stamens are most often normal. pi-5 flowers resemble those from 35S::SEP3 antisense plants. pi-5 contains missense mutation in the K domain (PIE125K). PIE125K exhibits defects in heterodimerization with its partner protein AP3. Via a reverse yeast two-hybrid screen, AP3K139E was isolated as a compensatory mutant of PIE125K. The compensatory interaction between PIE125K and AP3K139E is observed both in yeast two-hybrid assays and in planta. On its own, AP3K139E exhibits defects in specifying both petal and stamen identity. In addition, PIE125K is defective in interaction with SEPALLATA proteins in both two- and three-hybrid assays suggesting that PIE125K is defective in forming higher order complexes of MADS proteins. The decreased concentration of PI/AP3/SEP complexes offers an explanation for the petal defects observed in both pi-5 and 35S::SEP3 antisense plants.     

The K domain mediates heterodimerization of the Arabidopsis floral organ identity proteins,APETALA3 and PISTILLATA

MADS genes in plants encode key developmental regulators of vegetative and reproductive development. The majority of well-characterized plant MADS proteins contain two conserved domains, the DNA-binding MADS domain and the K domain. The K domain is predicted to form three amphipathic alpha-helices referred to as K1, K2, and K3. In this report, we define amino acids and subdomains important for heterodimerization between the two Arabidopsis floral organ identity MADS proteins APETALA3 (AP3) and PISTILLATA (PI). Analysis of mutants defective in dimerization demonstrates that K1, K2 and the region between K1 and K2 are critical for the strength of AP3/PI dimerization. The majority of the critical amino acids are hydrophobic indicating that the K domain mediates AP3/PI interaction primarily through hydrophobic interactions. Specially, K1 of AP3 and PI resembles a leucine zipper motif. Most mutants defective in AP3/PI heterodimerization in yeast exhibit partial floral organ identity function in transgenic Arabidopsis. Our results also indicate that the motif containing Asn-98 and specific charged residues in K1 (Glu-97 in PI and Arg-102 in AP3) are important for both the strength and specificity of AP3/PI heterodimer formation.     

Interactions between 'Candidatus Phytoplasma mali' and the apple endophyte Epicoccum nigrum in Catharanthus roseus plants

Aims: We investigated the ultrastructural and molecular interactions between ‘Candidatus Phytoplasma mali’ and the apple endophyte Epicoccum nigrum in the experimental host Catharanthus roseus to determine whether inoculation of endophyte could trigger defence reactions in the host. Methods and Results: Apple proliferation (AP) symptom severity was evaluated in AP‐grafted plants that were treated by E. nigrum and compared with untreated controls. Phytoplasma concentration was quantified by real‐time PCR in treated and untreated plants. Ultrastructural observations revealed that in endophyte‐treated periwinkles, modifications to phytoplasmas, such as irregular shape and cytoplasm confined to the periphery of the cell, and plant cytological changes, such as abundant callose depositions and P‐protein aggregations in the sieve elements, occurred. AP‐grafted plants that were treated by the endophyte (E. nigrum) showed a reduction in symptom severity; in particular, flowers appeared normal in shape and size, when compared with uninfected controls. Real‐time PCR indicated that phytoplasma concentration in AP‐grafted plants treated with E. nigrum was about 2·8 times lower than that in untreated ones. Conclusions: These results demonstrated that the inoculation with E. nigrum influenced phytoplasma infection in C. roseus plants; plant ultrastructural modifications allowed us to hypothesize an enhancing host defence response. Significance and Impact of the Study: At present, curative protocols against this phytoplasma are not available. Alternative approaches are thus required to reduce disease spread. Our study might represent a first step in the clarification of plant–phytoplasma–endophyte relationships to find possible strategies for the control of phytoplasma diseases.     

Separation of AG function in floral meristem determinacy from that in reproductive organ identity by expressing antisense AG RNA

The Arabidopsis floral homeotic gene AGAMOUS (AG) is a regulator of early flower development. The ag mutant phenotypes suggest that AG has two functions in flower development: (1) specifying the identity of stamens and carpels, and (2) controlling floral meristem determinacy. To dissect these two AG functions, we have generated transgenic Arabidopsis plants carrying an antisense AG construct. We found that all of the transgenic plants produced abnormal flowers, which can be classified into three types. Type I transgenic flowers are phenocopies of the ag-1 mutant flowers, with both floral meristem indeterminacy and floral organ conversion; type II flowers are indeterminate and have partial conversion of the reproductive organs; and type III flowers have normal stamens and carpels, but still have an indeterminate floral meristem inside the fourth whorl of fused carpels. The existence of type III flowers indicates that AG function can be perturbed to affect only floral meristem determinacy, but not floral organ identity. Furthermore, the fact that floral meristem determinacy is affected in all transformants, but floral organ identity only in a subset of them, suggests that the former may required a higher level of AG activity than the latter. This hypothesis is supported by the levels of AG'mRNA detected in different transformants with different frequencies of distinct types of abnormal antisense AG transgenic flowers. Finally, since AG inhibits the expression of another floral regulatory gene AP1, we examined AP1 expression in antisense AG flowers, and found that AP1 is expressed at a relatively high level in the center of type II flowers, but very little or below detectable levels in the inner whorls of type III flowers. These results provide further insights into the interaction of AG and AP1 and how such an interaction may control both organ identity and floral meristem determinacy.     

Euscelis incisus (Cicadellidae,Deltocephalinae), a natural vector of 16SrIII‐B phytoplasma causing multiple inflorescence disease of Cirsium arvense

We investigated multiple inflorescence disease of Cirsium arvense (CMI) and its association with phytoplasmas of the 16SrIII‐B subgroup, potential natural vector(s) and reservoir plant(s). From five locations in northern Serbia, 27 plants of C. arvense, 1 C. vulgare and 3 Carduus acanthoides with symptoms of multiple inflorescences (MIs) were collected and tested for 16SrIII group phytoplasmas. All symptomatic plants were found to be infected. Tentative reservoir plants and insect vectors were collected at a Dobanovci site where the continuous presence of CMI disease was recorded. Among the 19 most abundant plant species submitted to phytoplasma testing, all symptomless, the presence of the 16SrIII group was detected only in two legumes: Lathyrus tuberosus (2/5) and L. aphaca (1/5). Among 19 insect species from six families of Auchenorrhyncha, the deltocephalid leafhopper Euscelis incisus was the only insect carrying a 16SrIII phytoplasma (10% of analysed individuals). Transmission trials were performed with naturally infected E. incisus adults of the summer generation and with a laboratory population reared on red clover. After an acquisition period of 48 h on C. arvense symptomatic for MIs and a latent period of 28 days, 83% of the E. incisus adults (300/360) were infected with CMI phytoplasma. In two transmission tests, the leafhoppers successfully transmitted the phytoplasma to exposed plants (C. arvense and periwinkle), proving its role as a natural vector. Test plants of C. arvense infected with the 16SrIII‐B phytoplasma expressed typical symptoms similar to those observed in the field, such as MIs or the absence of flowering, shortened internodes and plant desiccation. Typical symptoms in infected periwinkles were virescence and phyllody. The molecular characterisation of the CMI phytoplasma isolates from diseased and asymptomatic field‐collected plants, vectors, and test plants was performed by sequence analyses of the 16S rRNA, rpl22‐rps3 and rpl15‐secY genes. Phylogenetic analyses of other members of the 16SrIII group of phytoplasmas indicated closest relatedness with clover yellow edge phytoplasma (CYE) of the 16SrIII‐B subgroup.     

Distance-based measurement determines the coexistence of B protein hetero- and homodimers in lily tepal and stamen tetrameric complexes

The floral quartet model proposes that plant MADS box proteins function as higher order tetrameric complexes. However, in planta evidence for MADS box tetramers remains scarce. Here, we applied a strategy using in vivo fluorescence resonance energy transfer (FRET) based on the distance change and distance symmetry of stable tetrameric complexes in tobacco (Nicotiana benthamiana) leaf cells to improve the accuracy of the estimation of heterotetrameric complex formation. This measuring system precisely verified the stable state of Arabidopsis petal (AP3/PI/SEP3/AP1) and stamen (AP3/PI/SEP3/AG) complexes and showed that the lily (Lilium longiflorum) PI co-orthologs LMADS8 and LMADS9 likely formed heterotetrameric petal complexes with Arabidopsis AP3/SEP3/AP1, which rescued petal defects of pi mutants. However, L8/L9 did not form heterotetrameric stamen complexes with Arabidopsis AP3/SEP3/AG to rescue the stamen defects of the pi mutants. Importantly, this system was applied successfully to find complicated tepal and stamen heterotetrameric complexes in lily. We found that heterodimers of B function AP3/PI orthologs (L1/L8) likely coexist with the homodimers of PI orthologs (L8/L8, L9/L9) to form five (two most stable and three stable) tepal- and four (one most stable and three stable) stamen-related heterotetrameric complexes with A/E and C/E function proteins in lily. Among these combinations, L1 preferentially interacted with L8 to form the most stable heterotetrameric complexes, and the importance of the L8/L8 and L9/L9 homodimers in tepal/stamen formation in lily likely decreased to a minor part during evolution. The system provides substantial improvements for successfully estimating the existence of unknown tetrameric complexes in plants.