The effector SJP3 interferes with pistil development by sustaining SHORT VEGETATIVE PHASE 3 expression in jujube.

Jujube witches' broom (JWB) is a phytoplasma disease that causes severe damage to jujube (Ziziphus jujuba) crops worldwide. Diseased jujube plants show enhanced vegetative growth after floral reversion, including leafy flower structures (phyllody) and the fourth whorl converting into a vegetative shoot. In previous research, secreted JWB protein 3 (SJP3) was identified as an inducer of phyllody. However, the molecular mechanisms of SJP3-mediated pistil reversion remain unknown. Here, the effector SJP3 was found to interact with the MADS-box protein SHORT VEGETATIVE PHASE 3 (ZjSVP3). ZjSVP3 was expressed in young leaves and during the initial flower bud differentiation of healthy jujube-bearing shoots but was constitutively expressed in JWB phytoplasma-infected flowers until the later stage of floral development. The SJP3 effector showed the same expression pattern in the diseased buds and promoted ZjSVP3 accumulation in SJP3 transgenic jujube calli. The N-terminal domains of ZjSVP3 contributed to its escape from protein degradation in the presence of SJP3. Heterologous expression of ZjSVP3 in Nicotiana benthamiana produced typical pistil abnormalities, including trichome-enriched style and stem-like structures within the leaf-like ovary, which were consistent with those in the mildly malformed lines overexpressing SJP3. Furthermore, ectopic expression of ZjSVP3 directly bound to the zinc finger protein 8 (ZjZFP8) and MADS-box gene SHATTERPROOF 1 (ZjSHP1) promoters to regulate their expression, resulting in abnormal pistil development. Overall, effector SJP3-mediated derepression of ZjSVP3 sustained its expression to interfere with pistil development, providing insight into the mechanisms of pistil reversion caused by JWB phytoplasma in specific perennial woody plant species.

The 'Candidatus phytoplasma ziziphi' effectors SJP1 and SJP2 destabilise the bifunctional regulator ZjTCP7 to modulate floral transition and shoot branching.

Phytoplasmic SAP11 effectors alter host plant architecture and flowering time. However, the exact mechanisms have yet to be elucidated. Two SAP11-like effectors, SJP1 and SJP2, from 'Candidatus Phytoplasma ziziphi' induce shoot branching proliferation. Here, the transcription factor ZjTCP7 was identified as a central target of these two effectors to regulate floral transition and shoot branching. Ectopic expression of ZjTCP7 resulted in enhanced bolting and earlier flowering than did the control. Interaction and expression assays demonstrated that ZjTCP7 interacted with the ZjFT-ZjFD module, thereby enhancing the ability of these genes to directly bind to the ZjAP1 promoter. The effectors SJP1 and SJP2 unravelled the florigen activation complex by specifically destabilising ZjTCP7 and ZjFD to delay floral initiation. Moreover, the shoot branching of the ZjTCP7-SRDX transgenic Arabidopsis lines were comparable to those of the SJP1/2 lines, suggesting the involvement of ZjTCP7 in the regulation of shoot branching. ZjTCP7 interacted with the branching repressor ZjBRC1 to enhance suppression of the auxin efflux carrier ZjPIN3 expression. ZjTCP7 also directly bound to and upregulated the auxin biosynthesis gene ZjYUCCA2, thereby promoting auxin accumulation. Our findings confirm that ZjTCP7 serves as a bifunctional regulator destabilised by the effectors SJP1 and SJP2 to modulate plant development.

The 'Candidatus Phytoplasma ziziphi' effectors SJP1/2 negatively control leaf size by stabilizing the transcription factor ZjTCP2 in jujube.

Phytoplasmas manipulate host plant development to benefit insect vector colonization and their own invasion. However, the virulence factors and mechanisms underlying small-leaf formation caused by jujube witches' broom (JWB) phytoplasmas remain largely unknown. Here, effectors SJP1 and SJP2 from JWB phytoplasmas were identified to induce small-leaf formation in jujube (Ziziphus jujuba). In vivo interaction and expression assays showed that SJP1 and SJP2 interacted with and stabilized the transcription factor ZjTCP2. Overexpression of SJP1 and SJP2 in jujube induced ZjTCP2 accumulation. In addition, the abundance of miRNA319f_1 was significantly reduced in leaves of SJP1 and SJP2 transgenic jujube plants and showed the opposite pattern to the expression of its target, ZjTCP2, which was consistent with the pattern in diseased leaves. Overexpression of ZjTCP2 in Arabidopsis promoted ectopic leaves arising from the adaxial side of cotyledons and reduced leaf size. Constitutive expression of the miRNA319f_1 precursor in the 35S::ZjTCP2 background reduced the abundance of ZjTCP2 mRNA and reversed the cotyledon and leaf defects in Arabidopsis. Therefore, these observations suggest that effectors SJP1 and SJP2 induced small-leaf formation, at least partly, by interacting with and activating ZjTCP2 expression both at the transcriptional and the protein level, providing new insights into small-leaf formation caused by phytoplasmas in woody plants.

Jujube witches' broom phytoplasma effectors SJP1 and SJP2 induce lateral bud outgrowth by repressing the ZjBRC1-controlled auxin efflux channel.

Comprehensively controlling phytoplasma-associated jujube witches' broom (JWB) disease is extremely challenging for the jujube industry. Although the pathogenesis of phytoplasma disease has been highlighted in many plant species, the release of lateral buds from dormancy under JWB phytoplasma infection has not been characterized in woody perennial jujube. Here, two 16SrV-B group phytoplasma effectors, SJP1 and SJP2, were experimentally determined to induce witches' broom with increased lateral branches. In vivo interaction and subcellular localization analyses showed that both SJP1 and SJP2 were translocated from the cytoplasm to the nucleus to target the CYC/TB1-TCP transcription factor ZjBRC1. The N- and C-terminal coiled-coil domains of SJP1 and SJP2 were required for the TCP-binding ability. ZjBRC1 bound directly to the auxin efflux carrier ZjPIN1c/3 promoters and down-regulated their expression to promote the accumulation of endogenous auxin indole-3-acetic acid in jujube calli. Furthermore, JWB phytoplasma infection suppressed ZjBRC1 accumulation and induced ZjPIN1c/3 expression to stimulate lateral bud outgrowth. Therefore, SJP1 and SJP2 stimulate lateral bud outgrowth, at least partly, by repressing the ZjBRC1-controlled auxin efflux channel in jujube, representing a potential strategy for comprehensive phytoplasma-associated disease control and a resource for gene editing breeding to create new cultivars with varying degrees of shoot branching.

Insertion of a solo LTR retrotransposon associates with spur mutations in 'Red Delicious' apple (Malus × domestica).

KEY MESSAGE: Insertion of a solo LTR, which possesses strong bidirectional, stem-specific promoter activities, is associated with the evolution of a dwarfing apple spur mutation. Spur mutations in apple scions revolutionized global apple production. Since long terminal repeat (LTR) retrotransposons are tightly related to natural mutations, inter-retrotransposon-amplified polymorphism technique and genome walking were used to find sequences in the apple genome based on these LTRs. In 'Red Delicious' spur mutants, a novel, 2190-bp insertion was identified as a spur-specific, solo LTR (sLTR) located at the 1038th nucleotide of another sLTR, which was 1536 bp in length. This insertion-within-an-insertion was localized within a preexisting Gypsy-50 retrotransposon at position 3,762,767 on chromosome 4. The analysis of transcriptional activity of the two sLTRs (the 2190- and 1536-bp inserts) indicated that the 2190-bp sLTR is a promoter, capable of bidirectional transcription. GUS expression in the 2190-bp-sense and 2190-bp-antisense transgenic lines was prominent in stems. In contrast, no promoter activity from either the sense or the antisense strand of the 1536-bp sLTR was detected. From ~150 kb of DNA on each side of the 2190 bp, sLTR insertion site, corresponding to 300 kb of the 'Golden Delicious' genome, 23 genes were predicted. Ten genes had predicted functions that could affect shoot development. This first report, of a sLTR insertion associated with the evolution of apple spur mutation, will facilitate apple breeding, cloning of spur-related genes, and discovery of mechanisms behind dwarf habit.

Jujube witches' broom phytoplasmas inhibit ZjBRC1-mediated abscisic acid metabolism to induce shoot proliferation.

Jujube witches' broom (JWB) phytoplasmas parasitize the sieve tubes of diseased phloem and cause an excessive proliferation of axillary shoots from dormant lateral buds to favour their transmission. In previous research, two JWB effectors, SJP1 and SJP2, were identified to induce lateral bud outgrowth by disrupting ZjBRC1-mediated auxin flux. However, the pathogenesis of JWB disease remains largely unknown. Here, tissue-specific transcriptional reprogramming was examined to gain insight into the genetic mechanisms acting inside jujube lateral buds under JWB phytoplasma infection. JWB phytoplasmas modulated a series of plant signalling networks involved in lateral bud development and defence, including auxin, abscisic acid (ABA), ethylene, jasmonic acid, and salicylic acid. JWB-induced bud outgrowth was accompanied by downregulation of ABA synthesis within lateral buds. ABA application rescued the bushy appearances of transgenic Arabidopsis overexpressing SJP1 and SJP2 in Col-0 and ZjBRC1 in the brc1-2 mutant. Furthermore, the expression of ZjBRC1 and ABA-related genes ZjHB40 and ZjNCED3 was negatively correlated with lateral main bud outgrowth in decapitated healthy jujube. Molecular evidence showed that ZjBRC1 interacted with ZjBRC2 via its N-terminus to activate ZjHB40 and ZjNCED3 expression and ABA accumulation in transgenic jujube calli. In addition, ZjBRC1 widely regulated differentially expressed genes related to ABA homeostasis and ABA signalling, especially by binding to and suppressing ABA receptors. Therefore, these results suggest that JWB phytoplasmas hijack the ZjBRC1-mediated ABA pathways to stimulate lateral bud outgrowth and expansion, providing a strategy to engineer plants resistant to JWB phytoplasma disease and regulate woody plant architecture to promote crop yield and quality.

Identification, expression and miRNA targeting of auxin response factor genes related to phyllody in the witches' broom disease of jujube.

Jujube witches' broom (JWB) disease, associated with the presence of phytoplasmas, induces huge crop losses in the woody perennial fruit tree Ziziphus jujuba. An imbalance in the phytohormone auxin is thought to be a key factor in the development of the witches' broom symptoms, and in the alteration of floral development into leafy structures, termed phyllody. The Auxin Response Factor (ARF) gene family controls auxin-responsive gene expression during plant growth and development. However, it remains unknown if the ARF genes are involved in the formation of leaf-like flowers. In the present study, sixteen jujube ARF genes were identified bioinformatically and annotated based on the Z. jujuba cv. Dongzao genome. The ZjARFs were homologous to 12 out of the 23 Arabidopsis ARFs and were distributed in 8 jujube chromosomes and 3 unmapped scaffolds. Phylogenetic analysis grouped the ZjARFs into three classes. Spatio-temporal expression analysis revealed that the ZjARF genes were differentially expressed among different tissues during normal development. The expression of seven ZjARF genes was significantly decreased from flower buds to flowering. JWB-infected jujube plants developed the typical phyllody symptoms and showed lower auxin accumulation during floral development. ZjARF1, ZjARF2, ZjARF3, ZjARF4 and ZjARF8 resulted differentially regulated after phytoplasma infection. ZjARF4 was down-regulated before and during floral development in phytoplasma-infected plants, but it was significantly up-regulated before flowering and down-regulated during flowering in the healthy plants. Target site analysis showed that miRNA167, miRNA529 and miRNA2950 could directly target ZjARF4. Together, the data showed that the auxin-controlled ARF4 gene is likely involved in the disruption of floral development in phytoplasma-infected jujube plants.