Multi-Omics Approaches Provide New Insights into the Identification of Putative Fungal Effectors from Valsa mali.

Pathogenic fungi secrete numerous effectors into host cells to manipulate plants' defense mechanisms. Valsa mali, a necrotrophic fungus, severely impacts apple production in China due to the occurrence of Valsa canker. Here, we predicted 210 candidate effector protein (CEP)-encoding genes from V. mali. The transcriptome analysis revealed that 146 CEP-encoding genes were differentially expressed during the infection of the host, Malus sieversii. Proteome analysis showed that 27 CEPs were differentially regulated during the infection stages. Overall, 25 of the 146 differentially expressed CEP-encoding genes were randomly selected to be transiently expressed in Nicotiana benthamiana. Pathogenicity analysis showed that the transient expression of VM1G-05058 suppressed BAX-triggered cell death while the expression of VM1G-10148 and VM1G-00140 caused cell death in N. benthamiana. In conclusion, by using multi-omics analysis, we identified potential effector candidates for further evaluation in vivo. Our results will provide new insights into the investigation of virulent mechanisms of V. mali.

An efficient screening system of disease-resistant genes from wild apple, Malus sieversii in response to Valsa mali pathogenic fungus.

For molecular breeding of future apples, wild apple (Malus sieversii), the primary progenitor of domesticated apples, provides abundant genetic diversity and disease-resistance traits. Valsa canker (caused by the fungal pathogen Valsa mali) poses a major threat to wild apple population as well as to cultivated apple production in China. In the present study, we developed an efficient system for screening disease-resistant genes of M. sieversii in response to V. mali. An optimal agrobacterium-mediated transient transformation of M. sieversii was first used to manipulate in situ the expression of candidate genes. After that, the pathogen V. mali was inoculated on transformed leaves and stems, and 3 additional methods for slower disease courses were developed for V. mali inoculation. To identify the resistant genes, a series of experiments were performed including morphological (incidence, lesion area/length, fungal biomass), physiological (H2O2 content, malondialdehyde content), and molecular (Real-time quantitative Polymerase Chain Reaction) approaches. Using the optimized system, we identified two transcription factors with high resistance to V. mali, MsbHLH41 and MsEIL3. Furthermore, 35 and 45 downstream genes of MsbHLH41 and MsEIL3 were identified by screening the V. mali response gene database in M. sieversii, respectively. Overall, these results indicate that the disease-resistant gene screening system has a wide range of applications for identifying resistant genes and exploring their immune regulatory networks.

Genome-wide characterization and functional identification of MYB genes in Malus sieversii infected by Valsa mali.

Among the most important transcription factors in plants, the v-myb avian myeloblastosis viral oncogene homolog (MYB) regulates the expression network of response genes under stresses such as fungal infection. In China, the canker disease Valsa mali threatens the survival of Malus sieversii, an ancestor of cultivated apples. Using the M. sieversii genome, we identified 457 MsMYB and 128 R2R3-MsMYB genes that were randomly distributed across 17 chromosomes. Based on protein sequence and structure, the R2R3-MsMYB genes were phylogenetically divided into 29 categories, and 26 conserved motifs were identified. We further predicted cis-elements in the 2000-kb promoter region of R2R3-MsMYBs based on the genome. Transcriptome analysis of M. sieversii under V. mali infection showed that 27 R2R3-MsMYBs were significantly differentially expressed, indicating their key role in the response to V. mali infection. Using transient transformation, MsMYB14, MsMYB24, MsMYB39, MsMYB78, and MsMYB108, which were strongly induced by V. mali infection, were functionally identified. Among the five MsMYBs, MsMYB14 and MsMYB78 were both important in enhancing resistance to diseases, whereas MsMYB24 inhibited resistance. Based on the results of this study, we gained a better understanding of the MsMYB transcription factor family and laid the foundation for a future research program on disease prevention strategies in M. sieversii.

Genome-Wide Identification of bHLH Transcription Factor Family in Malus sieversii and Functional Exploration of MsbHLH155.1 Gene under Valsa Canker Infection.

Xinjiang wild apple (Malus sieversii) is an ancient relic; a plant with abundant genetic diversity and disease resistance. Several transcription factors were studied in response to different biotic and abiotic stresses on the wild apple. Basic/helix-loop-helix (bHLH) is a large plant transcription factor family that plays important roles in plant responses to various biotic and abiotic stresses and has been extensively studied in several plants. However, no study has yet been conducted on the bHLH gene in M. sieversii. Based on the genome of M. sieversii, 184 putative MsbHLH genes were identified, and their physicochemical properties were studied. MsbHLH covered 23 subfamilies and lacked two subfamily genes of Arabidopsis thaliana based on the widely used classification method. Moreover, MsbHLH exon-intron structures matched subfamily classification, as evidenced by the analysis of their protein motifs. The analysis of cis-acting elements revealed that many MsbHLH genes share stress- and hormone-related cis-regulatory elements. These MsbHLH transcription factors were found to be involved in plant defense responses based on the protein-protein interactions among the differentially expressed MsbHLHs. Furthermore, 94 MsbHLH genes were differentially expressed in response to pathogenic bacteria. The qRT-PCR results also showed differential expression of MsbHLH genes. To further verify the gene function of bHLH, our study used the transient transformation method to obtain the overexpressed MsbHLH155.1 transgenic plants and inoculated them. Under Valsa canker infection, the lesion phenotype and physiological and biochemical indexes indicated that the antioxidant capacity of plants could increase and reduce the damage caused by membrane peroxidation. This study provides detailed insights into the classification, gene structure, motifs, chromosome distribution, and gene expression of bHLH genes in M. sieversii and lays a foundation for a better understanding disease resistance in plants, as well as providing candidate genes for the development of M. sieversii resistance breeding.

Genome-wide characterization of the chitinase gene family in wild apple (Malus sieversii) and domesticated apple (Malus domestica) reveals its role in resistance to Valsa mali.

Chitinases are responsible for catalyzing the hydrolysis of chitin and contribute to plant defense against fungal pathogens by degrading fungal chitin. In this study, genome-wide identification of the chitinase gene family of wild apple (Malus sieversii) and domesticated apple (Malus domestica) was conducted, and the expression profile was analyzed in response to Valsa mali infection. A total of 36 and 47 chitinase genes belonging to the glycosyl hydrolase 18 (GH18) and 19 (GH19) families were identified in the genomes of M. sieversii and M. domestica, respectively. These genes were classified into five classes based on their phylogenetic relationships and conserved catalytic domains. The genes were randomly distributed on the chromosomes and exhibited expansion by tandem and segmental duplication. Eight of the 36 MsChi genes and 17 of the 47 MdChi genes were differentially expressed in response to V. mali inoculation. In particular, MsChi35 and its ortholog MdChi41, a class IV chitinase, were constitutively expressed at high levels in M. sieversii and domesticated apple, respectively, and may play a crucial role in the defense response against V. mali. These results improve knowledge of the chitinase gene family in apple species and provide a foundation for further studies of fungal disease prevention in apple.

Impact of Cadmium Stress on Growth and Physio-Biochemical Attributes of Eruca sativa Mill.

Plants may experience adverse effects from Cadmium (Cd). As a result of its toxicity and mobility within the soil-plant continuum, it is attracting the attention of soil scientists and plant nutritionists. In this study, we subjected young Eruca sativa Mill. seedlings to different levels of Cd applications (0, 1.5, 6 and 30 µmol/L) via pot experiment to explore its morpho-physio-biochemical adaptations. Our results revealed a significant Cd accumulation in leaves at high Cd stress. It was also demonstrated that Cd stress inhibited photosynthetic rate and pigment levels, ascorbate peroxidase (APX), guaiacol peroxidase (GPX), catalase (CAT), and superoxide dismutase (SOD) enzyme activities, and increased malondialdehyde (MDA) levels. Conversely, the concentration of total ascorbate (TAS) increased at all levels of Cd application, whereas that of ascorbic acid (ASA), and dehydroascorbate (DHA) increased at 1.5 (non-significant), 6, 30 and 6 µmol/L (significant), though their concentrations decreased non-significantly at 30 µmol/L application. In conclusion, Cd-subjected E. sativa seedlings diverted much energy from growth towards the synthesis of anti-oxidant metabolites and osmolytes. However, they did not seem to have protected the E. sativa seedlings from Cd-induced oxidative stress, causing a decrease in osmotic adjustment, and an increase in oxidative damage, which resulted in a reduction in photosynthesis and growth. Accordingly, we recommend that the cultivation of E. sativa should be avoided on soil with Cd contamination.

Transcriptome profiling of Malus sieversii under freezing stress after being cold-acclimated.

BACKGROUND: Freezing temperatures are an abiotic stress that has a serious impact on plant growth and development in temperate regions and even threatens plant survival. The wild apple tree (Malus sieversii) needs to undergo a cold acclimation process to enhance its freezing tolerance in winter. Changes that occur at the molecular level in response to low temperatures are poorly understood in wild apple trees. RESULTS: Phytohormone and physiology profiles and transcriptome analysis were used to elaborate on the dynamic response mechanism. We determined that JA, IAA, and ABA accumulated in the cold acclimation stage and decreased during freezing stress in response to freezing stress. To elucidate the molecular mechanisms of freezing stress after cold acclimation, we employed single molecular real-time (SMRT) and RNA-seq technologies to study genome-wide expression profiles in wild apple. Using the PacBio and Illumina platform, we obtained 20.79G subreads. These reads were assembled into 61,908 transcripts, and 24,716 differentially expressed transcripts were obtained. Among them, 4410 transcripts were differentially expressed during the whole process of freezing stress, and these were examined for enrichment via GO and KEGG analyses. Pathway analysis indicated that "plant hormone signal transduction", "starch and sucrose metabolism", "peroxisome" and "photosynthesis" might play a vital role in wild apple responses to freezing stress. Furthermore, the transcription factors DREB1/CBF, MYC2, WRKY70, WRKY71, MYB4 and MYB88 were strongly induced during the whole stress period. CONCLUSIONS: Our study presents a global survey of the transcriptome profiles of wild apple trees in dynamic response to freezing stress after two days cold acclimation and provides insights into the molecular mechanisms of freezing adaptation of wild apple plants for the first time. The study also provides valuable information for further research on the antifreezing reaction mechanism and genetic improvement of M. sieversii after cold acclimation.

PacBio full-length transcriptome of wild apple (Malus sieversii) provides insights into canker disease dynamic response.

BACKGROUND: Valsa canker is a serious disease in the stem of Malus sieversii, caused by Valsa mali. However, little is known about the global response mechanism in M. sieversii to V. mali infection. RESULTS: Phytohormone jasmonic acid (JA) and salicylic acid (SA) profiles and transcriptome analysis were used to elaborate on the dynamic response mechanism. We determined that the JA was initially produced to respond to the necrotrophic pathogen V. mali infection at the early response stage, then get synergistically transduced with SA to respond at the late response stage. Furthermore, we adopted Pacific Biosciences (PacBio) full-length sequencing to identify differentially expressed transcripts (DETs) during the canker response stage. We obtained 52,538 full-length transcripts, of which 8139 were DETs. Total 1336 lncRNAs, 23,737 alternative polyadenylation (APA) sites and 3780 putative transcription factors (TFs) were identified. Additionally, functional annotation analysis of DETs indicated that the wild apple response to the infection of V. mali involves plant-pathogen interaction, plant hormone signal transduction, flavonoid biosynthesis, and phenylpropanoid biosynthesis. The co-expression network of the differentially expressed TFs revealed 264 candidate TF transcripts. Among these candidates, the WRKY family was the most abundant. The MsWRKY7 and MsWRKY33 were highly correlated at the early response stage, and MsWRKY6, MsWRKY7, MsWRKY19, MsWRKY33, MsWRKY40, MsWRKY45, MsWRKY51, MsWRKY61, MsWRKY75 were highly correlated at the late stage. CONCLUSIONS: The full-length transcriptomic analysis revealed a series of immune responsive events in M. sieversii in response to V. mali infection. The phytohormone signal pathway regulatory played an important role in the response stage. Additionally, the enriched disease resistance pathways and differentially expressed TFs dynamics collectively contributed to the immune response. This study provides valuable insights into a dynamic response in M. sieversii upon the necrotrophic pathogen V. mali infection, facilitates understanding of response mechanisms to canker disease for apple, and provides supports in the identification of potential resistance genes in M. sieversii.

Reverse Chromatin Immunoprecipitation (R-ChIP) enables investigation of the upstream regulators of plant genes.

DNA binding proteins carry out important and diverse functions in the cell, including gene regulation, but identifying these proteins is technically challenging. In the present study, we developed a technique to capture DNA-associated proteins called reverse chromatin immunoprecipitation (R-ChIP). This technology uses a set of specific DNA probes labeled with biotin to isolate chromatin, and the DNA-associated proteins are then identified using mass spectrometry. Using R-ChIP, we identified 439 proteins that potentially bind to the promoter of the Arabidopsis thaliana gene AtCAT3 (AT1G20620). According to functional annotation, we randomly selected 5 transcription factors from these candidates, including bZIP1664, TEM1, bHLH106, BTF3, and HAT1, to verify whether they in fact bind to the AtCAT3 promoter. The binding of these 5 transcription factors was confirmed using chromatin immunoprecipitation quantitative real-time PCR and electrophoretic mobility shift assays. In addition, we improved the R-ChIP method using plants in which the DNA of interest had been transiently introduced, which does not require the T-DNA integration, and showed that this substantially improved the protein capture efficiency. These results together demonstrate that R-ChIP has a wide application to characterize chromatin composition and isolate upstream regulators of a specific gene.

Identification and characterization of cadmium stress-related LncRNAs from Betula platyphylla.

Cadmium (Cd) is one of the most serious global environmental pollutants, which inhibits plant growth and interferes with their physiological processes. However, there have been few studies on the involvement of long noncoding RNAs (lncRNAs) in Cd tolerance. In the present study, we identified the lncRNAs from Betula platyphylla (birch) that respond to Cd stress. Thirty lncRNAs that were differentially expressed under Cd treatment were identified, including 16 upregulated and 14 downregulated lncRNAs. Nine differentially regulated lncRNAs were selected for further characterization. These lncRNAs were transiently overexpressed in birch plants to determine their roles in Cd tolerance. Among them, two lncRNAs conferred Cd tolerance and two induced sensitivity to Cd stress. We further determined the Cd tolerance of four target genes of the lncRNAs involved in Cd tolerance, including l-lactate dehydrogenase A (LDHA),heat shock protein (HSP18.1), yellow stripe-like protein (YSL9), and H/ACA ribonucleoprotein complex subunit 2-like protein (HRCS2L). Among them, HSP18.1 and LDHA showed improved tolerance to Cd stress, whereas the other two genes did not appear to be involved in Cd tolerance. These results suggested that lncRNAs can up- or downregulate their target genes to improve Cd tolerance. These results increase our understanding of lncRNA-mediated Cd tolerance.

Betula platyphylla BpHOX2 transcription factor binds to different cis-acting elements and confers osmotic tolerance.

The homeodomain-leucine zipper (HD-Zip) proteins play crucial roles in plant developmental and environmental responses. However, how they mediate gene expression to facilitate abiotic stress tolerance remains unknown. In the present study, we characterized BpHOX2 (encoding a HD-Zip I family protein) from birch (Betula platyphylla). BpHOX2 is predominately expressed in mature stems and leaves, but expressed at a low level in apical buds and roots, suggesting that it has tissue-specific characteristics. BpHOX2 expression was highly induced by osmotic and salt, but only slightly induced by abscisic acid. Overexpression of BpHOX2 markedly improved osmotic tolerance, while knockdown of BpHOX2 increased sensitivity to osmotic stress. BpHOX2 could induce the expression of pyrroline-5-carboxylate synthase, peroxidase, and superoxide dismutase genes to improve proline levels and the reactive oxygen species scavenging capability. Chromatin immunoprecipitation sequencing combined with RNA sequencing showed that BpHOX2 could bind to at least four cis-acting elements, including dehydration-responsive element "RCCGAC", Myb-p binding box "CCWACC," and two novel cis-acting elements with the sequences of "AAGAAG" and "TACGTG" (termed HBS1 and HBS2, respectively) to regulate gene expression. Our results suggested that BpHOX2 is a transcription factor that binds to different cis-acting elements to regulate gene expression, ultimately improving osmotic tolerance in birch.

Building a Robust Chromatin Immunoprecipitation Method with Substantially Improved Efficiency.

Chromatin immunoprecipitation (ChIP) is the gold-standard method for detection of interactions between proteins and chromatin and is a powerful tool for identification of epigenetic modifications. Although ChIP protocols for plant species have been developed, many specific features of plants, especially woody plants, still hinder the efficiency of immunoprecipitation, resulting in inefficient ChIP enrichment and an active demand for a highly efficient ChIP protocol. In this study, using birch (Betula platyphylla) and Arabidopsis (Arabidopsis thaliana) as the research materials, we identified five factors closely associated with ChIP efficiency, including crosslinking, concentration of chromatin using centrifugal filters, use of a different immunoprecipitation buffer, rescue of DNA with proteinase K, and use of Suc to increase immunoprecipitation efficiency. Optimization of any these factors can significantly improve ChIP efficiency. Considering these factors together, we developed a robust ChIP protocol that achieved a 14-fold improvement in ChIP enrichment for birch and a >6-fold improvement for Arabidopsis compared to the standard ChIP method. As this ChIP method works well in both birch and Arabidopsis, it should also be suitable for other woody and herbaceous species. In addition, this ChIP method enables detection of low-abundance transcription factor-DNA interactions and may extend the application of ChIP in the plant kingdom.

Non-canonical AUX/IAA protein IAA33 competes with canonical AUX/IAA repressor IAA5 to negatively regulate auxin signaling.

The phytohormone auxin controls plant growth and development via TIR1-dependent protein degradation of canonical AUX/IAA proteins, which normally repress the activity of auxin response transcription factors (ARFs). IAA33 is a non-canonical AUX/IAA protein lacking a TIR1-binding domain, and its role in auxin signaling and plant development is not well understood. Here, we show that IAA33 maintains root distal stem cell identity and negatively regulates auxin signaling by interacting with ARF10 and ARF16. IAA33 competes with the canonical AUX/IAA repressor IAA5 for binding to ARF10/16 to protect them from IAA5-mediated inhibition. In contrast to auxin-dependent degradation of canonical AUX/IAA proteins, auxin stabilizes IAA33 protein via MITOGEN-ACTIVATED PROTEIN KINASE 14 (MPK14) and does not affect IAA33 gene expression. Taken together, this study provides insight into the molecular functions of non-canonical AUX/IAA proteins in auxin signaling transduction.

A Gene Regulatory Network Controlled by BpERF2 and BpMYB102 in Birch under Drought Conditions.

Gene expression profiles are powerful tools for investigating mechanisms of plant stress tolerance. Betula platyphylla (birch) is a widely distributed tree, but its drought-tolerance mechanism has been little studied. Using RNA-Seq, we identified 2917 birch genes involved in its response to drought stress. These drought-responsive genes include the late embryogenesis abundant (LEA) family, heat shock protein (HSP) family, water shortage-related and ROS-scavenging proteins, and many transcription factors (TFs). Among the drought-induced TFs, the ethylene responsive factor (ERF) and myeloblastosis oncogene (MYB) families were the most abundant. BpERF2 and BpMYB102, which were strongly induced by drought and had high transcription levels, were selected to study their regulatory networks. BpERF2 and BpMYB102 both played roles in enhancing drought tolerance in birch. Chromatin immunoprecipitation combined with qRT-PCR indicated that BpERF2 regulated genes such as those in the LEA and HSP families, while BpMYB102 regulated genes such as Pathogenesis-related Protein 1 (PRP1) and 4-Coumarate:Coenzyme A Ligase 10 (4CL10). Multiple genes were regulated by both BpERF2 and BpMYB102. We further characterized the function of some of these genes, and the genes that encode Root Primordium Defective 1 (RPD1), PRP1, 4CL10, LEA1, SOD5, and HSPs were found to be involved in drought tolerance. Therefore, our results suggest that BpERF2 and BpMYB102 serve as transcription factors that regulate a series of drought-tolerance genes in B. platyphylla to improve drought tolerance.

An Improved Syringe Agroinfiltration Protocol to Enhance Transformation Efficiency by Combinative Use of 5-Azacytidine, Ascorbate Acid and Tween-20.

Syringe infiltration is an important transient transformation method that is widely used in many molecular studies. Owing to the wide use of syringe agroinfiltration, it is important and necessary to improve its transformation efficiency. Here, we studied the factors influencing the transformation efficiency of syringe agroinfiltration. The pCAMBIA1301 was transformed into Nicotiana benthamiana leaves for investigation. The effects of 5-azacytidine (AzaC), Ascorbate acid (ASC) and Tween-20 on transformation were studied. The ß-glucuronidase (GUS) expression and GUS activity were respectively measured to determine the transformation efficiency. AzaC, ASC and Tween-20 all significantly affected the transformation efficiency of agroinfiltration, and the optimal concentrations of AzaC, ASC and Tween-20 for the transgene expression were identified. Our results showed that 20 µM AzaC, 0.56 mM ASC and 0.03% (v/v) Tween-20 is the optimal concentration that could significantly improve the transformation efficiency of agroinfiltration. Furthermore, a combined supplement of 20 µM AzaC, 0.56 mM ASC and 0.03% Tween-20 improves the expression of transgene better than any one factor alone, increasing the transgene expression by more than 6-fold. Thus, an optimized syringe agroinfiltration was developed here, which might be a powerful method in transient transformation analysis.