Unraveling a Small Secreted Peptide SUBPEP3 That Positively Regulates Salt-Stress Tolerance in Pyrus betulifolia.

Small secreted peptides (SSPs) play important roles in regulating plants' growth and development in response to external stimulus, but the genes and functions of SSPs in many species are still unknown. Therefore, it is particularly significant to characterize and annotate SSP genes in plant genomes. As a widely used stock of pears, Pyrus betulifolia has strong resistance to biotic and abiotic stresses. In this study, we analyzed the SSPs genes in the genome of P. betulifolia according to their characteristics and homology. A total of 1195 SSP genes were identified, and most of them are signaling molecules. Among these, we identified a new SSP, subtilase peptide 3 (SUBPEP3), which derived from the PA region of preSUBPEP3, increasing the expression level under salt stress. Both adding synthetic peptide SUBPEP3 to the culture medium of pears and the overexpression of SUBPEP3 in tobacco can improve the salt tolerance of plants. In summary, we annotated the SSP genes in the P. betulifolia genome and identified a small secreted peptide SUBPEP3 that regulates the salt tolerance of P. betulifolia, which provides an important theoretical basis for further revealing the function of SSPs.

The Mechanism of Ovule Abortion in Self-Pollinated 'Hanfu' Apple Fruits and Related Gene Screening.

Apples exhibit S-RNase-mediated self-incompatibility and typically require cross-pollination in nature. 'Hanfu' is a cultivar that produces abundant fruit after self-pollination, although it also shows a high rate of seed abortion afterwards, which greatly reduces fruit quality. In this study, we investigated the ovule development process and the mechanism of ovule abortion in apples after self-pollination. Using a DIC microscope and biomicroscope, we found that the abortion of apple ovules occurs before embryo formation and results from the failure of sperm-egg fusion. Further, we used laser-assisted microdissection (LAM) cutting and sperm and egg cell sequencing at different periods after pollination to obtain the genes related to ovule abortion. The top 40 differentially expressed genes (DEGs) were further verified, and the results were consistent with switching the mechanism at the 5' end of the RNA transcript (SMART-seq). Through this study, we can preliminarily clarify the mechanism of ovule abortion in self-pollinated apple fruits and provide a gene reserve for further study and improvement of 'Hanfu' apple fruit quality.

The microRNA miR482 regulates NBS-LRR genes in response to ALT1 infection in apple.

Plants are frequently attacked by a variety of pathogens and thus have evolved a series of defense mechanisms, one important mechanism is resistance gene (R gene)-mediated disease resistance, but its expression is tightly regulated. NBS-LRR genes are the largest gene family of R genes. microRNAs (miRNAs) target to a number of NBS-LRR genes and trigger the production of phased small interfering RNAs (phasiRNAs) from these transcripts. phasiRNAs cis or trans regulate NBS-LRR genes, which can result in the repression of R gene expression. In this study, we screened for upregulated miR482 in the susceptible apple cultivar 'Golden Delicious' (GD) after inoculation with the fungal pathogen Alternaria alternata f. sp. mali (ALT1). Additionally, through combined degradome sequencing, we identified a gene targeted by miR482, named MdTNL1, a gene encoding a TIR-NBS-LRR (Toll/interleukin1 receptor-nucleotide binding site-leucine-rich repeat) protein. This gene exhibited a significant down-regulation post ALT1 inoculation, suggesting an impact on gene expression mediated by miRNA regulation. miR482 could cleave MdTNL1 and generate phasiRNAs at the cleavage site. We found that overexpression of miR482 inhibited the expression of MdTNL1 and thus reduced the disease resistance of GD, while silencing of miR482 increased the expression of MdTNL1 and thus improved the disease resistance of GD. This work elucidates key mechanisms underlying the immune response to Alternaria infection in apple. Identification of the resistance genes involved will enable molecular breeding for prevention and control of Alternaria leaf spot disease in this important fruit crop.

Highly-Adaptable Optothermal Nanotweezers for Trapping, Sorting, and Assembling across Diverse Nanoparticles.

Optical manipulation of various kinds of nanoparticles is vital in biomedical engineering. However, classical optical approaches demand higher laser power and are constrained by diffraction limits, necessitating tailored trapping schemes for specific nanoparticles. They lack a universal and biocompatible tool to manipulate nanoparticles of diverse sizes, charges, and materials. Through precise modulation of diffusiophoresis and thermo-osmotic flows in the boundary layer of an optothermal-responsive gold film, highly adaptable optothermal nanotweezers (HAONTs) capable of manipulating a single nanoparticle as small as sub-10 nm are designed. Additionally, a novel optothermal doughnut-shaped vortex (DSV) trapping strategy is introduced, enabling a new mode of physical interaction between cells and nanoparticles. Furthermore, this versatile approach allows for the manipulation of nanoparticles in organic, inorganic, and biological forms. It also offers versatile function modes such as trapping, sorting, and assembling of nanoparticles. It is believed that this approach holds the potential to be a valuable tool in fields such as synthetic biology, optofluidics, nanophotonics, and colloidal science.

Early and Sensitive Detection of Pathogens for Public Health and Biosafety: An Example of Surveillance and Genotyping of SARS-CoV-2 in Sewage Water by Cas12a-Facilitated Portable Plasmonic Biosensor.

Infectious diseases severely threaten public health and global biosafety. In addition to transmission through the air, pathogenic microorganisms have also been detected in environmental liquid samples, such as sewage water. Conventional biochemical detection methodologies are time-consuming and cost-ineffective, and their detection limits hinder early diagnosis. In the present study, ultrafine plasmonic fiber probes with a diameter of 125 µm are fabricated for clustered regularly interspaced short palindromic repeats/CRISPR-associated protein (CRISPR/Cas)-12a-mediated sensing of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Single-stranded DNA exposed on the fiber surface is trans-cleaved by the Cas12a enzyme to release gold nanoparticles that are immobilized onto the fiber surface, causing a sharp reduction in the surface plasmon resonance (SPR) wavelength. The proposed fiber probe is virus-specific with the limit of detection of ~2,300 copies/ml, and genomic copy numbers can be reflected as shifts in wavelengths. A total of 21 sewage water samples have been examined, and the data obtained are consistent with those of quantitative polymerase chain reaction (qPCR). In addition, the Omicron variant and its mutation sites have been fast detected using S gene-specific Cas12a. This study provides an accurate and convenient approach for the real-time surveillance of microbial contamination in sewage water.

Ultrasensitive and Specific Clustered Regularly Interspaced Short Palindromic Repeats Empowered a Plasmonic Fiber Tip System for Amplification-Free Monkeypox Virus Detection and Genotyping.

The urgent necessity for highly sensitive diagnostic tools has been accentuated by the ongoing mpox (monkeypox) virus pandemic due to the complexity in identifying asymptomatic and presymptomatic carriers. Traditional polymerase chain reaction-based tests, despite their effectiveness, are hampered by limited specificity, expensive and bulky equipment, labor-intensive operations, and time-consuming procedures. In this study, we present a clustered regularly interspaced short palindromic repeats (CRISPR)/Cas12a-based diagnostic platform with a surface plasmon resonance-based fiber tip (CRISPR-SPR-FT) biosensor. The compact CRISPR-SPR-FT biosensor, with a 125 µm diameter, offers high stability and portability, enabling exceptional specificity for mpox diagnosis and precise identification of samples with a fatal mutation site (L108F) in the F8L gene. The CRISPR-SPR-FT system can analyze viral double-stranded DNA from mpox virus without amplification in under 1.5 h with a limit of detection below 5 aM in plasmids and about 59.5 copies/µL when in pseudovirus-spiked blood samples. Our CRISPR-SPR-FT biosensor thus offers fast, sensitive, portable, and accurate target nucleic acid sequence detection.

The ß-1,3-Glucanase Degrades Callose at Plasmodesmata to Facilitate the Transport of the Ribonucleoprotein Complex in Pyrus betulaefolia.

Grafting is widely used to improve the stress tolerance and the fruit yield of horticultural crops. Ribonucleoprotein complexes formed by mRNAs and proteins play critical roles in the communication between scions and stocks of grafted plants. In Pyrus betulaefolia, ankyrin was identified previously to promote the long-distance movement of the ribonucleoprotein complex(PbWoxT1-PbPTB3) by facilitating callose degradation at plasmodesmata. However, the mechanism of the ankyrin-mediated callose degradation remains elusive. In this study, we discovered a ß-1,3-glucanase (EC 3.2.1.39, PbPDBG) using ankyrin as a bait from plasmodesmata by co-immunoprecipitation and mass spectrometry. Ankyrin was required for the plasmodesmata-localization of PbPDBG. The grafting and bombardment experiments indicated that overexpressing PbPDBG resulted in decreased callose content at plasmodesmata, and thereby promoting the long-distance transport of the ribonucleoprotein complex. Altogether, our findings revealed that PbPDBG was the key factor in ankyrin-mediated callose degradation at plasmodesmata.

Long-distance transport of the pear HMGR1 mRNA via the phloem is associated with enhanced salt tolerance.

Grafting is the main asexual propagation method for horticultural crops and can enhance their resistance to biotic or abiotic stress. Many mRNAs can be transported over long distances through the graft union, however, the function of mobile mRNAs remains poorly understood. Here, we exploited lists of candidate mobile mRNAs harboring potential 5-methylcytosine (m5C) modification in pear (Pyrus betulaefolia). dCAPS RT-PCR and RT-PCR were employed to demonstrate the mobility of the 3-hydroxy-3-methylglutaryl-coenzyme A reductase1 (PbHMGR1) mRNA in grafted plants of both pear and tobacco (Nicotiana tabacum). Overexpressing PbHMGR1 in tobacco plants enhanced salt tolerance during seed germination. In addition, both histochemical staining and GUS expression analysis showed that PbHMGR1 could directly respond to salt stress. Furthermore, it was found that the relative abundance of PbHMGR1 increased in heterografted scion, which avoided serious damage under salt stress. Collectively, these findings established that PbHMGR1 mRNA could act as a salt-responsive signal and move through the graft union to enhance salt tolerance of scion, which might be used as a new plant breeding technique to improve resistance of scion through a stress-tolerant rootstock.

CRISPR-Cas13a-powered electrochemical biosensor for the detection of the L452R mutation in clinical samples of SARS-CoV-2 variants.

Since the end of 2019, a highly contagious disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has deprived numerous lives worldwide, called COVID-19. Up to date, omicron is the latest variant of concern, and BA.5 is replacing the BA.2 variant to become the main subtype rampaging worldwide. These subtypes harbor an L452R mutation, which increases their transmissibility among vaccinated people. Current methods for identifying SARS-CoV-2 variants are mainly based on polymerase chain reaction (PCR) followed by gene sequencing, making time-consuming processes and expensive instrumentation indispensable. In this study, we developed a rapid and ultrasensitive electrochemical biosensor to achieve the goals of high sensitivity, the ability of distinguishing the variants, and the direct detection of RNAs from viruses simultaneously. We used electrodes made of MXene-AuNP (gold nanoparticle) composites for improved sensitivity and the CRISPR/Cas13a system for high specificity in detecting the single-base L452R mutation in RNAs and clinical samples. Our biosensor will be an excellent supplement to the RT-qPCR method enabling the early diagnosis and quick distinguishment of SARS-CoV-2 Omicron BA.5 and BA.2 variants and more potential variants that might arise in the future.

A chaperonin containing T-complex polypeptide-1 facilitates the formation of the PbWoxT1-PbPTB3 ribonucleoprotein complex for long-distance RNA trafficking in Pyrus betulaefolia.

Numerous plant endogenous mRNAs move via phloem and thus affect the growth and development of long-distant organs. mRNAs are transported with RNA-binding proteins forming a ribonucleoprotein complex. However, it remains elusive how such RNP complex assembles and facilitates mRNA trafficking. Protease digestion and RNA immunoprecipitation were used to investigate the RNP assembly function of the complete Chaperonin Containing T-complex Polypeptide-1. In situ hybridization, hairy root transformation, microprojectile bombardment, and grafting experiments demonstrate the role of CCT complex in the transport of a PbWoxT1-PbPTB3 RNP complex in Pyrus betulaefolia. PbCCT5 silenced caused defective movement of GFP-PbPTB3 and GFP-PbWoxT1 from hairy roots to new leaves via the phloem. PbCCT5 is shown to interact with PbPTB3. PbCCT complex enhanced PbPTB3 stabilization and permitted assembly of PbWoxT1 and PbPTB3 into an RNP complex. Furthermore, silencing of individual CCT subunits inhibited the intercellular movement of GFP-PbPTB3 and long-distance trafficking of PbWoxT1 and PbPTB3 in grafted plants. Taken together, the CCT complex assembles PbPTB3 and PbWoxT1 into an RNP complex in the phloem in order to facilitate the long-distance trafficking of PbWoxT1 in P. betulaefolia. This study therefore provides important insights into the mechanism of RNP complex formation and transport.

Glioma diagnosis and therapy: Current challenges and nanomaterial-based solutions.

Glioma is often referred to as one of the most dreadful central nervous system (CNS)-specific tumors with rapidly-proliferating cancerous glial cells, accounting for nearly half of the brain tumors at an annual incidence rate of 30-80 per a million population. Although glioma treatment remains a significant challenge for researchers and clinicians, the rapid development of nanomedicine provides tremendous opportunities for long-term glioma therapy. However, several obstacles impede the development of novel therapeutics, such as the very tight blood-brain barrier (BBB), undesirable hypoxia, and complex tumor microenvironment (TME). Several efforts have been dedicated to exploring various nanoformulations for improving BBB permeation and precise tumor ablation to address these challenges. Initially, this article briefly introduces glioma classification and various pathogenic factors. Further, currently available therapeutic approaches are illustrated in detail, including traditional chemotherapy, radiotherapy, and surgical practices. Then, different innovative treatment strategies, such as tumor-treating fields, gene therapy, immunotherapy, and phototherapy, are emphasized. In conclusion, we summarize the article with interesting perspectives, providing suggestions for future glioma diagnosis and therapy improvement.

A long noncoding RNA HILinc1 enhances pear thermotolerance by stabilizing PbHILT1 transcripts through complementary base pairing.

As global warming intensifies, heat stress has become a major environmental constraint threatening crop production and quality worldwide. Here, we characterize Heat-induced long intergenic noncoding RNA 1 (HILinc1), a cytoplasm-enriched lincRNA that plays a key role in thermotolerance regulation of pear (Pyrus spp.). HILinc1 Target 1 (PbHILT1) which is the target transcript of HILinc1, was stabilized via complementary base pairing to upregulate its expression. PbHILT1 could bind to Heat shock transcription factor A1b (PbHSFA1b) to enhance its transcriptional activity, leading to the upregulation of a major downstream transcriptional regulator, Multiprotein bridging factor 1c (PbMBF1c), during heat response. Transient overexpressing of either HILinc1 or PbHILT1 increases thermotolerance in pear, while transient silencing of HILinc1 or PbHILT1 makes pear plants more heat sensitive. These findings provide evidences for a new regulatory mechanism by which HILinc1 facilitates PbHSFA1b activity and enhances pear thermotolerance through stabilizing PbHILT1 transcripts.

A CRISPR/Cas12a-empowered surface plasmon resonance platform for rapid and specific diagnosis of the Omicron variant of SARS-CoV-2.

The outbreak of the COVID-19 pandemic was partially due to the challenge of identifying asymptomatic and presymptomatic carriers of the virus, and thus highlights a strong motivation for diagnostics with high sensitivity that can be rapidly deployed. On the other hand, several concerning SARS-CoV-2 variants, including Omicron, are required to be identified as soon as the samples are identified as 'positive'. Unfortunately, a traditional PCR test does not allow their specific identification. Herein, for the first time, we have developed MOPCS (Methodologies of Photonic CRISPR Sensing), which combines an optical sensing technology-surface plasmon resonance (SPR) with the 'gene scissors' clustered regularly interspaced short palindromic repeat (CRISPR) technique to achieve both high sensitivity and specificity when it comes to measurement of viral variants. MOPCS is a low-cost, CRISPR/Cas12a-system-empowered SPR gene-detecting platform that can analyze viral RNA, without the need for amplification, within 38 min from sample input to results output, and achieve a limit of detection of 15 fM. MOPCS achieves a highly sensitive analysis of SARS-CoV-2, and mutations appear in variants B.1.617.2 (Delta), B.1.1.529 (Omicron) and BA.1 (a subtype of Omicron). This platform was also used to analyze some recently collected patient samples from a local outbreak in China, identified by the Centers for Disease Control and Prevention. This innovative CRISPR-empowered SPR platform will further contribute to the fast, sensitive and accurate detection of target nucleic acid sequences with single-base mutations.

PbANK facilitates the long-distance movement of the PbWoxT1-PbPTB3 RNP complex by degrading deposited callose.

Grafting horticultural crops can result in phenotypic changes in the grafted materials due to the movement of macromolecular signals, including RNAs and proteins, across the graft union; however, little is known about the composition of trafficking ribonucleoprotein (RNP) complexes or how these macromolecules are transported. Here, we used the core of PbPTB3-PbWoxT1 RNP complex, PbPTB3, as bait to screen Pyrus betulaefolia cDNA library for its interaction partners. We identified an ankyrin protein, PbANK, that interacts with PbPTB3 to facilitate its transport through the phloem alongside PbWoxT1 mRNA. Heterografting experiments showed that silencing PbANK in rootstock prevented the transport of PbPTB3 and PbWoxT1 mRNA from the rootstock to the scion. Similarly, heterologous grafting experiments demonstrated that PbANK itself cannot be transported over long distances through a graft union. Fluorescence microscopy showed that silencing ANK affected the intercellular diffusion of PbPTB3 and increased callose deposition at plasmodesmata. Collectively, these findings demonstrate that PbANK mediates the long-distance movement of PbPTB3 and PbWoxT1 by degrading callose to increase the efficiency of cell-to-cell movement.

Exogenous gibberellin treatment improves fruit quality in self-pollinated apple.

Although a few apple (Malus × ×domestica) varieties are self-compatible, little is known about the differences in fruit quality between self- and cross-pollinated apple. In our current study, we compared the fruit quality of self-pollinated apple plants (cultivar 'Hanfu') in self-pollination or cross-pollinated by another cultivar 'Qinguan'. Analysis of fruit quality revealed substantial differences in the external qualities between self- and cross-pollinated apple fruit, but not in the internal qualities. Fruits harvested from self-pollinated 'Hanfu' were smaller and more asymmetrical than those harvested from the cross-pollinated plants. We developed a mathematical model describing how seed number and distribution affect fruit growth. According to this model, the fewer the seeds, the greater the force released from the seeds and the more asymmetrical the fruit. Detection of endogenous hormone and the associated gene expression revealed that gibberellin (GA) levels and GA transporter gene expression on the seedless side were significantly lower than those on the seeded side. Analysis of fruit pectin methylesterase activity and demethylated pectin levels indicated that the lack of GA limits fruit cell wall extension. Additionally, spraying the self-pollinating plants with gibberellic acid increased the fruit weight and lowered the proportion of asymmetrical fruit, recovering the exterior fruit quality to that of the cross-pollinated fruit. Furthermore, exogenous GA treatment increased the wax layer thickness and reduced the fruit water loss rate, leading to a dramatic improvement in fruit storage capacity. Therefore, exogenous GA treatment could be used to ensure regular fruit production of self-pollinated 'Hanfu'.

Polyamines Involved in Regulating Self-Incompatibility in Apple.

Apple exhibits typical gametophytic self-incompatibility, in which self-S-RNase can arrest pollen tube growth, leading to failure of fertilization. To date, there have been few studies on how to resist the toxicity of self-S-RNase. In this study, pollen tube polyamines were found to respond to self-S-RNase and help pollen tubes defend against self-S-RNase. In particular, the contents of putrescine, spermidine, and spermine in the pollen tube treated with self-S-RNase were substantially lower than those treated with non-self-S-RNase. Further analysis of gene expression of key enzymes in the synthesis and degradation pathways of polyamines found that the expression of DIAMINE OXIDASE 4 (MdDAO4) as well as several polyamine oxidases such as POLYAMINE OXIDASES 3 (MdPAO3), POLYAMINE OXIDASES 4 (MdPAO4), and POLYAMINE OXIDASES 6 (MdPAO6) were significantly up-regulated under self-S-RNase treatment, resulting in the reduction of polyamines. Silencing MdPAO6 in pollen tubes alleviates the inhibitory effect of self-S-RNase on pollen tube growth. In addition, exogenous polyamines also enhance pollen tube resistance to self-S-RNase. Transcriptome sequencing data found that polyamines may communicate with S-RNase through the calcium signal pathway, thereby regulating the growth of the pollen tubes. To summarize, our results suggested that polyamines responded to the self-incompatibility reaction and could enhance pollen tube tolerance to S-RNase, thus providing a potential way to break self-incompatibility in apple.

Two pathogenesis-related proteins interact with leucine-rich repeat proteins to promote Alternaria leaf spot resistance in apple.

Alternaria leaf spot in apple (Malus x domestica), caused by the fungal pathogen Alternaria alternata f. sp. mali (also called A. mali), is a devastating disease resulting in substantial economic losses. We previously established that the resistance (R) protein MdRNL2, containing a coiled-coil, nucleotide-binding, and leucine-rich repeat (CCR-NB-LRR) domain, interacts with another CCR-NB-LRR protein, MdRNL6, to form a MdRNL2-MdRNL6 complex that confers resistance to A. mali. Here, to investigate the function of the MdRNL2-MdRNL6 complex, we identified two novel pathogenesis-related (PR) proteins, MdPR10-1 and MdPR10-2, that interact with MdRNL2. Yeast two-hybrid (Y2H) assays and bimolecular fluorescence complementation (BiFC) assays confirmed that MdPR10-1 and MdPR10-2 interact with MdRNL2 and MdRNL6 at the leucine-rich repeat domain. Transient expression assays demonstrated that accumulation of MdPR10-1 and MdPR10-2 enhanced the resistance of apple to four strains of A. mali that we tested: ALT1, GBYB2, BXSB5, and BXSB7. In vitro antifungal activity assays demonstrated that both the proteins contribute to Alternaria leaf spot resistance by inhibiting fungal growth. Our data provide evidence for a novel regulatory mechanism in which MdRNL2 and MdRNL6 interact with MdPR10-1 and MdPR10-2 to inhibit fungal growth, thereby contributing to Alternaria leaf spot resistance in apple. The identification of these two novel PR proteins will facilitate breeding for fungal disease resistance in apple.

CCR -NB-LRR proteins MdRNL2 and MdRNL6 interact physically to confer broad-spectrum fungal resistance in apple (Malus × domestica).

Apple leaf spot, a disease caused by Alternaria alternata f. sp. mali and other fungal species, leads to severe defoliation and results in tremendous losses to the apple (Malus × domestica) industry in China. We previously identified three RPW8, nucleotide-binding, and leucine-rich repeat domain CCR -NB-LRR proteins (RNLs), named MdRNL1, MdRNL2, and MdRNL3, that contribute to Alternaria leaf spot (ALT1) resistance in apple. However, the role of NB-LRR proteins in resistance to fungal diseases in apple remains poorly understood. We therefore used MdRNL1/2/3 as baits to screen ALT1-inoculated leaves for interacting proteins and identified only MdRNL6 (another RNL) as an interactor of MdRNL2. Protein interaction assays demonstrated that MdRNL2 and MdRNL6 interact through their NB-ARC domains. Transient expression assays in apple indicated that complexes containing both MdRNL2 and MdRNL6 are necessary for resistance to Alternaria leaf spot. Intriguingly, the same complexes were also required to confer resistance to Glomerella leaf spot and Marssonina leaf spot in transient expression assays. Furthermore, stable transgenic apple plants with suppressed expression of MdRNL6 showed hypersensitivity to Alternaria leaf spot, Glomerella leaf spot, and Marssonina leaf spot; these effects were similar to the effects of suppressing MdRNL2 expression in transgenic apple plantlets. The identification of these novel broad-spectrum fungal resistance genes will facilitate breeding for fungal disease resistance in apple.

2D materials for bone therapy.

Due to their prominent physicochemical properties, 2D materials are broadly applied in biomedicine. Currently, 2D materials have achieved great success in treating many diseases such as cancer and tissue engineering as well as bone therapy. Based on their different characteristics, 2D materials could function in various ways in different bone diseases. Herein, the application of 2D materials in bone tissue engineering, joint lubrication, infection of orthopedic implants, bone tumors, and osteoarthritis are firstly reviewed comprehensively together. Meanwhile, different mechanisms by which 2D materials function in each disease reviewed below are also reviewed in detail, which in turn reveals the versatile functions and application of 2D materials. At last, the outlook on how to further broaden applications of 2D materials in bone therapies based on their excellent properties is also discussed.

The apple MdPTI1L kinase is phosphorylated by MdOXI1 during S-RNase-induced reactive oxygen species signaling in pollen tubes.

Apple (Malus domestica) exhibits classic S-RNase-mediated gametophytic self-incompatibility. Previous studies have shown that the S-RNase secreted from style cells could trigger signal transduction and defense responses mediated by Ca2+ and reactive oxygen species (ROS) after entering into the pollen tube. In this study, we investigated the downstream genes activated by ROS during S-RNase-mediated gametophytic self-incompatibility in pollen tubes. A substantial increase in ROS, as well as up-regulated expression of a serine-threonine protein kinase gene, OXIDATIVE SIGNAL-INDUCIBLE1 (MdOXI1), was detected in apple pollen tubes treated with self-S-RNase. A kinase assay-linked phosphoproteomics (KALIP) analysis suggested that MdOXI1 could bind and phosphorylate the downstream protein kinase Pto-interacting protein 1-like (MdPTI1L). The phosphorylation level of MdPTI1L was significantly reduced after silencing MdOXI1 with antisense oligonucleotides in the pollen tube. Silencing of either MdOXI1 or MdPTI1L alleviated the inhibitory effect of self-S-RNase on pollen tube growth. Our results thus indicate that MdPTI1L is phosphorylated by MdOXI1 in the pollen tube and participates in the ROS signaling pathway triggered by S-RNase.