Valsa mali PR1-like protein modulates an apple valine-glutamine protein to suppress JA signaling-mediated immunity.

Apple Valsa canker (AVC) is a devastating disease of apple (Malus × domestica), caused by Valsa mali (Vm). The Cysteine-rich secretory protein, Antigen 5, and Pathogenesis-related protein 1 (CAP) superfamily protein PATHOGENESIS-RELATED PROTEIN 1-LIKE PROTEIN c (VmPR1c) plays an important role in the pathogenicity of Vm. However, the mechanisms through which it exerts its virulence function in Vm-apple interactions remain unclear. In this study, we identified an apple valine-glutamine (VQ)-motif-containing protein, MdVQ29, as a VmPR1c target protein. MdVQ29-overexpressing transgenic apple plants showed substantially enhanced AVC resistance as compared with the wild type. MdVQ29 interacted with the transcription factor MdWRKY23, which was further shown to bind to the promoter of the jasmonic acid (JA) signaling-related gene CORONATINE INSENSITIVE 1 (MdCOI1) and activate its expression to activate the JA signaling pathway. Disease evaluation in lesion areas on infected leaves showed that MdVQ29 positively modulated apple resistance in a MdWRKY23-dependent manner. Furthermore, MdVQ29 promoted the transcriptional activity of MdWRKY23 toward MdCOI1. In addition, VmPR1c suppressed the MdVQ29-enhanced transcriptional activation activity of MdWRKY23 by promoting the degradation of MdVQ29 and inhibiting MdVQ29 expression and the MdVQ29-MdWRKY23 interaction, thereby interfering with the JA signaling pathway and facilitating Vm infection. Overall, our results demonstrate that VmPR1c targets MdVQ29 to manipulate the JA signaling pathway to regulate immunity. Thus, this study provides an important theoretical basis and guidance for mining and utilizing disease-resistance genetic resources for genetically improving apples.

Identification of an SCF Ubiquitin Ligase Complex that Contributes to Resistance Against Valsa Canker in Apple.

E3 ubiquitin ligases play a critical role in plant disease resistance. Among them, the Skp1-Cullin-F-box protein (SCF) ubiquitin ligase complex is the largest family and regulates the ubiquitination of a wide range of proteins. Apple Valsa canker (AVC) is a fungal disease of apple trees caused by the fungus Valsa mali, which can lead to significant economic losses. However, the function of the SCF complex in apple resistance to this disease is still largely unknown. In this study, we identified an SCF ubiquitin ligase complex that can enhance resistance to Valsa canker in apple. Disease evaluation experiments demonstrated that MdSkp1 increased apple resistance to AVC. Furthermore, MdSkp1 interacted with an F-box protein, MdSKIP14, and interacted with a cullin-1 protein, MdCUL1, to form an SCF ubiquitin ligase complex. Additionally, we revealed both MdSKIP14 and MdCUL1 as positive regulators of AVC resistance. In conclusion, our results identified an SCF complex capable of contributing to apple resistance against AVC, providing a theoretical basis for apple disease resistance and the sustainable development of the industry. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

A fungal microRNA-like RNA subverts host immunity and facilitates pathogen infection by silencing two host receptor-like kinase genes.

Small RNAs (sRNAs) play important roles in various biological processes by silencing their corresponding target genes in most eukaryotes. However, cross-kingdom regulation mediated by fungal microRNA-like RNAs (milRNAs) in plant-pathogen interactions is still largely unknown. Using molecular, genetic, histological, and biochemical approaches, we found that the apple tree Valsa canker pathogen Valsa mali milRNA Vm-milR1 could suppress the host immunity by silencing two host receptor-like kinase genes, MdRLKT1 and MdRLKT2. Vm-milR1 was highly induced during V. mali infection. Deletion of Vm-milR1 precursor abolished the generation of Vm-milR1 and reduced the virulence of V. mali. Inoculation of Vm-milR1 deletion mutants induced the host defence responses, including reactive oxygen species (ROS) accumulation, callose deposition, and high expression of defence-related genes. Furthermore, Vm-milR1 was confirmed to be able to suppress the expression of MdRLKT1 and MdRLKT2 in a sequence-specific manner. Moreover, overexpression of either MdRLKT1 or MdRLKT2 enhanced apple resistance to V. mali by activating the host defence responses. Furthermore, knockdown of MdRLKT1 or MdRLKT2 compromised the host resistance to V. mali. Our study revealed that V. mali was equipped with Vm-milR1 as an sRNA effector to silence host receptor-like kinase genes, suppress the host defence responses, and facilitate pathogen infection.

The Apple Receptor-Like Kinase MdSRLK3 Positively Regulates Resistance Against Pathogenic Fungus Valsa mali by Affecting the Ca2+ Signaling Pathway.

Valsa mali is the main pathogenic fungus that causes the apple Valsa canker, a destructive disease severely threatening apple production in the world. However, the underlying key components involved in resistance against V. mali in apple trees remain largely unexplored. Here, we isolated and functionally characterized a G-type lectin S-receptor-like protein kinase MdSRLK3 from the cultivar Royal Gala derivative line GL-3. qRT-PCR showed that the relative expression of MdSRLK3 in apple branches reached its highest level at 24 h post V. mali inoculation, which was 13.42 times higher than without inoculation. Transient overexpression of MdSRLK3 enhanced apple resistance against V. mali, while transient silencing of MdSRLK3 reduced its resistance against the pathogen. More importantly, stable silencing of MdSRLK3 resulted in reduced resistance against this fungus. Furthermore, we demonstrated that MdSRLK3 positively regulated apple resistance by affecting the Ca2+ signaling pathway, and the regulation was also related to the H2O2 and callose signaling pathways. Overall, our data reveal that MdSRLK3 is a positive regulator of apple immunity.

Auxin regulates anthocyanin biosynthesis through the auxin repressor protein MdIAA26.

Auxin plays an important role in plant growth and development; for example, it regulates the elongation and division of plant cells, the formation of plantlet's geotropism and phototropism, and the growth of main lateral roots and hypocotyl. IAA gene is associated with auxin and can response to biotic and abiotic stress in plants. However, the regulatory effect of auxin on anthocyanin accumulation has been rarely reported. In this study, we show that auxin inhibites the accumulation of anthocyanin and decreases the expression of genes related to anthocyanin synthesis in calli, leaves, and seedlings of apple. The expression levels of MdIAA family genes were determined, and we found that MdIAA26 significantly responded to auxin, which also induced MdIAA26 degradation. Functional analysis of MdIAA26 showed that overexpressing MdIAA26 in apple calli and Arabidopsis could promote the accumulation of anthocyanin and up-regulate the genes related to anthocyanin synthesis. Furthermore, the MdIAA26-overexpressing Arabidopsis could counteract auxin-induced inhibition on anthocyanin accumulation, which indicates that auxin inhibits the accumulation of anthocyanin in apple by degrading MdIAA26 protein.

BTB-BACK Domain E3 Ligase MdPOB1 Suppresses Plant Pathogen Defense against Botryosphaeria dothidea by Ubiquitinating and Degrading MdPUB29 Protein in Apple.

Apple ring rot is a severe disease that affects the yield and quality of apple fruits worldwide. However, the underlying molecular mechanism that involved in this process still remains largely unexplored. Here, we report that apple POZ/BTB CONTAINING-PROTEIN 1 (MdPOB1), a BTB-BACK domain E3 ligase protein, functions to suppress apple pathogen defense against Botryosphaeria dothidea (B. dothidea). Both in vitro and in vivo assays indicated that MdPOB1 interacted directly with and degraded apple U-box E3 ligase MdPUB29, a well-established positive regulator of plant innate immunity, through the ubiquitin/26S proteasome pathway. A series of transgenic analyses in apple fruits demonstrated that MdPOB1 affected apple pathogen defense against B. dothidea at least partially, if not completely, via regulating MdPUB29. Additionally, it was found that the apple pathogen defense against B. dothidea was correlated with the H2O2 contents and the relative expression of salicylic acid (SA) synthesis- and SA signaling-related genes, which might be regulated via degradation of MdPUB29 by MdPOB1. Overall, our findings provide new insights into the mechanism of the MdPOB1 modulation of apple ring rot resistance, which occur by directly regulating potential downstream target protein MdPUB29 for proteasomal degradation in apple.

The apple U-box E3 ubiquitin ligase MdPUB29 contributes to activate plant immune response to the fungal pathogen Botryosphaeria dothidea.

MAIN CONCLUSION: MdPUB29 is a positive regulator of the defense response to the fungal pathogen Botryosphaeria dothidea possibly by directly regulating the salicylic acid (SA) content as well as SA synthesis-related and signaling-related gene transcription. In plants, ubiquitin E3 ligases containing a U-box domain (PUBs, Plant U-box E3 ubiquitin ligase) have been identified as key regulators of fundamental cellular processes, such as cellular growth, development, and apoptosis, as well as biotic and abiotic stress responses. However, the function of PUBs in apple ring rot remains elusive. Here, we isolated the U-box E3 ligase MdPUB29 from the apple cultivar 'Royal Gala' and characterized its function in plant pathogen defense against Botryosphaeria dothidea. qRT-PCR showed that the expression of MdPUB29 was significantly induced in apple fruits after B. dothidea infection. Overexpression of the MdPUB29 gene in apple calli increased the resistance to B. dothidea infection. In contrast, silencing MdPUB29 in apple calli resulted in reduced resistance. Ectopic expression of MdPUB29 in Arabidopsis also exhibited enhanced resistance to B. dothidea infection compared to that of the wild-type (Col) control. In addition, it was found that the increase of plant pathogen defense was correlated with the increased salicylic acid (SA) content, as well as SA synthesis-related and signaling-related gene transcription in comparison to the wild type. We elucidated the mechanism by which MdPUB29 elevates plant pathogen defense against B. dothidea possibly by regulating the SA pathway.

The regulatory module MdPUB29-MdbHLH3 connects ethylene biosynthesis with fruit quality in apple.

The plant hormone ethylene is critical for climacteric fruit ripening, while glucose and anthocyanins determine the fruit quality of climacteric fruits such as apple. Understanding the exact molecular mechanism for this process is important for elucidating the interconnection of ethylene and fruit quality. Overexpression of apple MdbHLH3 gene, an anthocyanin-related basic helix-loop-helix transcription factor (bHLH TF) gene, promotes ethylene production, and transgenic apple plantlets and trees exhibit ethylene-related root developmental abnormalities, premature leaf senescence, and fruit ripening. Biochemical analyses demonstrate that MdbHLH3 binds to the promoters of three genes that are involved in ethylene biosynthesis, including MdACO1, MdACS1, and MdACS5A, activating their transcriptional expression, thereby promoting ethylene biosynthesis. High glucose-inhibited U-box-type E3 ubiquitin ligase MdPUB29, the ortholog of Arabidopsis AtPUB29 in apple, influences the expression of ethylene biosynthetic genes and ethylene production by direct ubiquitination of the MdbHLH3 protein. Our findings provide new insights into the ubiquitination of MdbHLH3 by glucose-inhibited ubiquitin E3 ligase MdPUB29 in the regulation of ethylene biosynthesis as well as indicate that the regulatory module MdPUB29-MdbHLH3 connects ethylene biosynthesis with fruit quality in apple.

An Apple Protein Kinase MdSnRK1.1 Interacts with MdCAIP1 to Regulate ABA Sensitivity.

ABA is a crucial phytohormone for development and stress responses in plants. Snf1-related protein kinase 1.1 (SnRK1.1) is involved in the ABA response. However, the molecular mechanism underlying the SnRK1.1 response to ABA is largely unknown. Here, it was found that overexpression of the apple MdSnRK1.1 gene enhanced ABA sensitivity in both transgenic apple calli and Arabidopsis seedlings. Subsequently, a yeast two-hybrid screen demonstrated that MdCAIP1 (C2-domain ABA Insensitive Protein1) interacted with MdSnRK1.1. Their interaction was further confirmed by pull-down and co-immunoprecipitation assays. Expression of the MdCAIP1 gene was positively induced by ABA. Its overexpression enhanced ABA sensitivity in transgenic apple calli. Furthermore, it was found that MdSnRK1.1 phosphorylated the MdCAIP1 protein in vivo and promoted its degradation in vitro and in vivo. As a result, MdSnRK1.1 inhibited MdCAIP1-mediated ABA sensitivity, and MdCAIP1 partially reduced MdSnRK1.1-mediated ABA sensitivity. Our findings indicate that MdSnRK1.1 plays an important role in the ABA response, partially by controlling the stability of the MdCAIP1 protein.

MdVQ12 confers resistance to Valsa mali by regulating MdHDA19 expression in apple.

Valine-glutamine (VQ) motif-containing proteins play a crucial role in plant biotic stress responses. Apple Valsa canker, caused by the ascomycete Valsa mali, stands as one of the most severe diseases affecting apple trees. Nonetheless, the underlying resistance mechanism of VQ proteins against this disease has remained largely unexplored. This study reports MdVQ12, a VQ motif-containing protein, as a positive regulator of apple Valsa canker resistance. Genetic transformation experiments demonstrated that MdVQ12 overexpression increased resistance to V. mali, while gene silencing lines exhibited significantly reduced resistance. MdVQ12 interacted with the transcription factor MdWRKY23, which bound to the promoter of the histone deacetylase gene MdHDA19, activating its expression. MdHDA19 enhanced apple resistance to V. mali by participating in the jasmonic acid (JA) and ethylene (ET) signalling pathways. Additionally, MdVQ12 promoted the transcriptional activity of MdWRKY23 towards MdHDA19. Our findings reveal that MdVQ12 enhances apple resistance to V. mali by regulating MdHDA19 expression and thereby regulating the JA and ET signalling pathways, offering potential candidate gene resources for breeding apple Valsa canker-resistant germplasm.

Apple ethylene response factor MdERF11 confers resistance to fungal pathogen Botryosphaeria dothidea.

Ethylene response factor (ERF) is a plant-specific transcription factor involved in many biological processes including root formation, hypocotyl elongation, fruit ripening, organ senescence and stress responses, as well as fruit quality formation. However, its underlying mechanism in plant pathogen defense against Botryosphaeria dothidea (B. dothidea) remains poorly understood. Here, we isolate MdERF11, an apple nucleus-localized ERF transcription factor, from apple cultivar 'Royal Gala'. qRT-PCR assays show that the expression of MdERF11 is significantly induced in apple fruits after B. dothidea infection. Overexpression of MdERF11 gene in apple calli significantly increases the resistance to B.dothidea infection, while silencing MdERF11 in apple calli results in reduced resistance. Ectopic expression of MdERF11 in Arabidopsis also exhibits enhanced resistance to B. dothidea infection compared to that of wild type. Infections in apple calli and Arabidopsis leaves by B. dothidea respectively cause an increase in endogenous levels of salicylic acid (SA) followed by induction of SA synthesis-related and signaling-related gene expression. Taken together, these findings illustrate a potential mechanism by which MdERF11 elevates plant pathogen defense against B. dothidea by regulating SA synthesis pathway.