Transcription factor PbbZIP4 is targeted for proteasome-mediated degradation by the ubiquitin ligase PbATL18 to influence pear's resistance to Colletotrichum fructicola by regulating the expression of PbNPR3.

Pear anthracnose caused by Colletotrichum fructicola is one of the main fungal diseases in all pear-producing areas. The degradation of ubiquitinated proteins by the 26S proteasome is a regulatory mechanism of eukaryotes. E3 ubiquitin ligase is substrate specific and is one of the most diversified and abundant enzymes in the regulation mechanism of plant ubiquitination. Although numerous studies in other plants have shown that the degradation of ubiquitinated proteins by the 26S proteasome is closely related to plant immunity, there are limited studies on them in pear trees. Here, we found that an E3 ubiquitin ligase, PbATL18, interacts with and ubiquitinates the transcription factor PbbZIP4, and this process is enhanced by C. fructicola infection. PbATL18 overexpression in pear callus enhanced resistance to C. fructicola infection, whereas PbbZIP4 overexpression increased sensitivity to C. fructicola infection. Silencing PbATL18 and PbbZIP4 in Pyrus betulaefolia seedlings resulted in opposite effects, with PbbZIP4 silencing enhancing resistance to C. fructicola infection and PbATL18 silencing increasing sensitivity to C. fructicola infection. Using yeast one-hybrid screens, an electrophoretic mobility shift assay, and dual-luciferase assays, we demonstrated that the transcription factor PbbZIP4 upregulated the expression of PbNPR3 by directly binding to its promoter. PbNPR3 is one of the key genes in the salicylic acid (SA) signal transduction pathway that can inhibit SA signal transduction. Here, we proposed a PbATL18-PbbZIP4-PbNPR3-SA model for plant response to C. fructicola infection. PbbZIP4 was ubiquitinated by PbATL18 and degraded by the 26S proteasome, which decreased the expression of PbNPR3 and promoted SA signal transduction, thereby enhancing plant C. fructicola resistance. Our study provides new insights into the molecular mechanism of pear response to C. fructicola infection, which can serve as a theoretical basis for breeding superior disease-resistant pear varieties.

PbHsfC1a-coordinates ABA biosynthesis and H2O2 signalling pathways to improve drought tolerance in Pyrus betulaefolia.

Abiotic stresses have had a substantial impact on fruit crop output and quality. Plants have evolved an efficient immune system to combat abiotic stress, which employs reactive oxygen species (ROS) to activate the downstream defence response signals. Although an aquaporin protein encoded by PbPIP1;4 is identified from transcriptome analysis of Pyrus betulaefolia plants under drought treatments, little attention has been paid to the role of PIP and ROS in responding to abiotic stresses in pear plants. In this study, we discovered that overexpression of PbPIP1;4 in pear callus improved tolerance to oxidative and osmotic stresses by reconstructing redox homeostasis and ABA signal pathways. PbPIP1;4 overexpression enhanced the transport of H2O2 into pear and yeast cells. Overexpression of PbPIP1;4 in Arabidopsis plants mitigates the stress effects caused by adding ABA, including stomatal closure and reduction of seed germination and seedling growth. Overexpression of PbPIP1;4 in Arabidopsis plants decreases drought-induced leaf withering. The PbPIP1;4 promoter could be bound and activated by TF PbHsfC1a. Overexpression of PbHsfC1a in Arabidopsis plants rescued the leaf from wilting under drought stress. PbHsfC1a could bind to and activate AtNCED4 and PbNCED4 promoters, but the activation could be inhibited by adding ABA. Besides, PbNCED expression was up-regulated under H2O2 treatment but down-regulated under ABA treatment. In conclusion, this study revealed that PbHsfC1a is a positive regulator of abiotic stress, by targeting PbPIP1;4 and PbNCED4 promoters and activating their expression to mediate redox homeostasis and ABA biosynthesis. It provides valuable information for breeding drought-resistant pear cultivars through gene modification.

Genome-wide identification of the mitogen-activated protein kinase kinase kinase (MAPKKK) in pear (Pyrus bretschneideri) and their functional analysis in response to black spot.

MAIN CONCLUSION: Identification of MAPKKK genes in pear and functional characterization of PbrMAPKKK82 in response to pear black spot. Mitogen-activated protein kinase kinase kinase (MAPKKK) is located upstream of the MAPK cascade pathway. This region senses extracellular stimuli via the signaling molecule or by themselves and is activated by phosphorylation. In this study, we identified 108 PbrMAPKKK genes from the pear genome. The genes were divided into three subfamilies and contained the conserved domain. Except for chromosome 7, there were 93 PbrMAPKKK genes randomly distributed on 16 out of the 17 chromosomes, while 15 PbrMAPKKK genes were detected on unknown chromosomes. They largely originated from whole-genome duplication (WGD) and dispersed events. In the expression analysis of PbrMAPKKK genes in seven pear tissue types by using a database, 20 PbrMAPKKK genes were selected to verify the expression associated with different resistance in two varieties by quantitative real-time PCR (qRT-PCR). The results showed that PbrMAPKKK12, PbrMAPKKK13, PbrMAPKKK53, PbrMAPKKK60, PbrMAPKKK65, PbrMAPKKK82, PbrMAPKKK83, and PbrMAPKKK96 were correlated with black spot resistance. PbrMAPKKK3, PbrMAPKKK9, PbrMAPKKK11, PbrMAPKKK34, PbrMAPKKK80, PbrMAPKKK81, PbrMAPKKK99, and PbrMAPKKK100 were correlated with black spot susceptibility, while the PbrMAPKKK gene positively responded to the life process of pear resistance to black spot. Furthermore, virus-induced gene silencing (VIGS) indicated that the PbrMAPKKK82 gene enhanced resistance to pear black spot disease.

PuNDH9, a subunit of ETC Complex I regulates plant defense by interacting with PuPR1.

NAD+ and NADH play critical roles in energy metabolism, cell death, and gene expression. The NADH-ubiquinone oxidoreductase complex (Complex I) has been long known as a key enzyme in NAD+ and NADH metabolism. In the present study, we found and analyzed a new subunit of Complex I (NDH9), which was isolated from Pyrus ussuriensis combined with RT-PCR. Following infection with A. alternata, RT-qPCR analysis demonstrated an increase in the expression of PuNDH9. Genetic manipulation of PuNDH9 levels suggested that PuNDH9 plays key roles in NADH/NAD+ homeostasis, defense enzyme activities, ROS generation, cell death, gene expression, energy metabolism, and mitochondrial functions during the pear- A. alternata interaction. Furthermore, Y2H, GST-pull down, and a split-luciferase complementation imaging assays revealed that PuNDH9 interacts with PuPR1. We discover that PuNDH9 and PuPR1 synergistically activate defense enzyme activities, ROS accumulation, cell death, and plant defenses. Collectively, our findings reveal that PuNDH9 is likely important for plant defenses.

PbWRKY18 promotes resistance against black spot disease by activation of the chalcone synthase gene PbCHS3 in pear.

Flavonoids are plant pigments that play a major role in plant defense and have significant health benefits to humans. Chalcone synthase (CHS) is an important enzyme in flavonoid biosynthesis and investigation transcription factors (TFs) regulating its expression and downstream targets is critical to understanding its mechanism. Here, a novel TF, PbWRKY18, was isolated from the pear Pyrus betulaefolia. Its expression was evaluated in various tissues by RT-PCR, particularly in response to Alternaria alternata, the pathogen responsible for black spot disease, and exogenous hormone administration. The PbWRKY18 protein was primarily found in the nucleus where it regulated transcriptional activity. Yeast one-hybrid and dual-luciferase reporter assays showed a strong association between PbWRKY18 and the PbCHS3 promoter, which drives PbCHS3 expression. It was also found that PbCHS3 was critical for the development of resistance against black spot disease. In addition, PbWRKY18 was found to significantly increase the expression of PbCHS3 and salicylic acid-related genes, as well as defense enzyme activity and tolerance to black spot disease. PbWRKY18 or PbCHS3 knockdown in pear attenuates resistance to Alternaria alternata. In summary, the study identified a novel WRKY18-CHS3 axis involved in resistance against black spot disease in pear.

The PbbHLH62/PbVHA-B1 module confers salt tolerance through modulating intracellular Na+/K+ homeostasis and reactive oxygen species removal in pear.

The vacuolar H+-ATPase (V-ATPase) is a multi-subunit membrane protein complex, which plays pivotal roles in building up an electrochemical H+-gradient across tonoplast, energizing Na+ sequestration into the central vacuole, and enhancing salt stress tolerance in plants. In this study, a B subunit of V-ATPase gene, PbVHA-B1 was discovered and isolated from stress-induced P. betulaefolia combining with RT-PCR method. The RT-qPCR analysis revealed that the expression level of PbVHA-B1 was upregulated by salt, drought, cold, and exogenous ABA treatment. Subcellular localization analyses showed that PbVHA-B1 was located in the cytoplasm and nucleus. Moreover, overexpression of PbVHA-B1 gene noticeably increased the ATPase activity and the tolerance to salt in transgenic Arabidopsis plants. In contrast, knockdown of PbVHA-B1 gene in P.betulaefolia by virus-induced gene silencing had reduced resistance to salt stress. In addition, using yeast one-hybride (Y1H) and yeast two-hybride (Y2H) screens, PbbHLH62, a bHLH transcription factor, was identified as a partner of the PbVHA-B1 promoter and protein. Then, we also found that PbbHLH62 positively regulate the expression of PbVHA-B1 and the ATPase activity after salt stress treatment. These findings provide evidence that PbbHLH62 played a critical role in the salt response. Collectively, our results demonstrate that a PbbHLH62/PbVHA-B1 module plays a positive role in salt tolerance by maintain intracellular ion and ROS homeostasis in pear.

Pyrus betulaefolia ERF3 interacts with HsfC1a to coordinately regulate aquaporin PIP1;4 and NCED4 for drought tolerance.

Environmental disasters like drought reduce agricultural output and plant growth. Redox management significantly affects plant stress responses. An earlier study found that PbPIP1;4 transports H2O2 and promotes H2O2 downstream cascade signaling to restore redox equilibrium. However, this regulatory mechanism requires additional investigation. In this search, the AP2 domain-containing transcription factor was isolated by screening Y1H from the wild pear (Pyrus betulaefolia) cDNA library, named PbERF3. The overexpression of PbERF3 in pear callus and Arabidopsis enhanced plant resistance to drought and re-established redox balance. The transcripts of the NCEDs gene were upregulated under drought stress. The drought stress-related abscisic acid (ABA) signaling pathway modulates PbERF3. PbERF3 silencing lowered drought tolerance. Furthermore, yeast 2-hybrid, luciferase, bimolecular fluorescence complementation, and co-immunoprecipitation assays verified that PbERF3 physically interacted with PbHsfC1a. The PbERF3-PbHsfC1a heterodimer coordinately bound to PbPIP1;4 and PbNCED4 promoter, therefore activating both the H2O2 and the ABA signaling pathway. This work revealed a novel PbERF3-PbHsfC1a-PbNCED4-PbPIP1;4 regulatory module, in which PbERF3 interacts with PbHsfC1a to trigger the expression of target genes. This module establishes an interaction between the H2O2 signaling component PbPIP1;4 and the ABA pathways component PbNCED4, enabling a response to drought.

PbrWRKY70 increases pear (Pyrus bretschneideri Rehd) black spot disease tolerance by negatively regulating ethylene synthesis via PbrERF1B-2.

Various pear plant cultivars exhibit diverse abilities to resist pear black spot disease (BSD), while the precise molecular mechanisms of resistance against pear BSD remain unclear. This study proposed a profound expression of a WRKY gene, namely PbrWRKY70, derived from Pyrus bretschneideri Rehd, within a BSD-resistant pear cultivar. Comparative analysis against the wild-type revealed that the overexpression of PbrWRKY70 engendered augmented BSD resistance of transgenic Arabidopsis thaliana and pear calli. Notably, the transgenic plants exhibited higher activities of superoxide dismutase and peroxidase, along with an elevated capacity to counteract superoxide anions via increased anti-O2-. Additionally, these plants displayed diminished lesion diameter, as well as reduced levels of hydrogen peroxide, malondialdehyde and 1-aminocyclopropane-1-carboxylic acid (ACC) contents. We subsequently demonstrated that PbrWRKY70 selectively bound to the promoter region of ethylene-responsive transcription factor 1B-2 (PbrERF1B-2), a potential negative regulator of ACC, thereby downregulating the expression of ACC synthase gene (PbrACS3). Consequently, we confirmed that PbrWRKY70 could enhance pear resistance against BSD by reducing ethylene production via modulation of the PbrERF1B-2-PbrACS3 pathway. This study established the pivotal relationship among PbrWRKY70, ethylene synthesis and pear BSD resistance, fostering the development of novel BSD-resistant cultivars. Furthermore, this breakthrough holds the potential to enhance pear fruit yield and optimize storage and processing during the later stages of fruit maturation.