The F-box protein OsFBX156 positively regulates rice defence against the blast fungus Magnaporthe oryzae by mediating ubiquitination-dependent degradation of OsHSP71.1.

F-box protein is a subunit of the SCF (SKP1-CUL1-F-box protein) E3 ubiquitin ligase complex, which plays a critical role in regulating different pathways in plant immunity. In this study, we identified the rice (Oryza sativa) F-box protein OsFBX156, which targets the heat shock protein 70 (OsHSP71.1) to regulate resistance to the rice blast fungus Magnaporthe oryzae. Overexpression of OsFBX156 or knockout of OsHSP71.1 in rice resulted in the elevation of pathogenesis-related (PR) genes and an induction burst of reactive oxygen species (ROS) after flg22 and chitin treatments, thereby enhancing resistance to M. oryzae. Furthermore, OsFBX156 can promote the degradation of OsHSP71.1 through the 26S proteasome pathway. This study sheds lights on a novel mechanism wherein the F-box protein OsFBX156 targets OsHSP71.1 for degradation to promote ROS production and PR gene expression, thereby positively regulating rice innate immunity.

Arabidopsis Tubby domain-containing F-box proteins positively regulate immunity by modulating PI4Kß protein levels.

The Tubby domain, named after the TUBBY protein in mice, binds to phosphatidylinositol 4,5-bisphosphate. Arabidopsis has 11 Tubby domain-containing proteins referred to as Tubby-Like Proteins (TLPs). Of the 11 TLPs, 10 possess the N-terminal F-box domain, which can interact with SKP-like proteins and form SKP1-Cullin-F-box E3 ligase complexes. Although mice TUBBY has been extensively studied, plant TLPs' functions are scarcely detailed. In this study, we show that the Arabidopsis Tubby-like protein 6 (TLP6) and its redundant homologs, TLP1, TLP2, TLP5, and TLP10, positively regulate Arabidopsis immune responses. Furthermore, in an immunoprecipitation mass spectrometry analysis to search for ubiquitination substrates of the TLPs, we identified two redundant phosphoinositide biosynthesis enzymes, phosphatidylinositol 4-kinase ß proteins (PI4Kßs), PI4Kß1 and PI4Kß2, as TLP interactors. Importantly, TLP6 overexpression lines fully phenocopy the phenotypes of the pi4kß1,2 mutant, while TLP6 overexpression also leads to increased PI4Kß2 ubiquitination and reduction in its protein level in a proteasome-dependent manner. Most significantly, TLP6 overexpression does not further enhance the autoimmunity of the pi4kß1,2 double mutant, supporting the hypothesis that TLP6 targets the PI4Kßs for ubiquitination and degradation. Thus, our study reveals a novel mechanism where TLPs promote plant immune responses by modulating the PI4Kßs protein levels.

Complete mitochondrial genome sequencing and identification of candidate genes responsible for C5-type cytoplasmic male sterility in cabbage (B. oleracea var. capitata).

Cytoplasmic male sterility (CMS) is widely used in cruciferous vegetables hybrid breeding. The C5-type CMS cabbage line exhibits stable male sterility and offers great value for cabbage breeding. However, the underlying CMS mechanism remains unclear. Here, the complete mitochondrial genome was sequenced and assembled for this line. The genome size was 221,862 bp. Mitochondrial genome comparison showed that the mitochondrial genome was likely generated by recombination with a nap-type CMS B. napus strain. Sixty-seven unknown-function open reading frames (ORFs) were identified. Seven orfs, orf114a, orf123a, orf188a, orf222a, orf261a, orf286a, and orf322a, were specifically identified in this genome. The presence of these candidate CMS genes decreased ATPase activity and ATP content by affecting the transcript levels of energy metabolism-related genes and F1F0-ATP synthase assembly. Among them, orf188a, orf222a, orf261a, orf286a, and orf322a possessed a transmembrane structure, and orf188a was cotranscribed with rps7 and trnfM. orf222a was partially homologous to atp8 and coexpressed with nad5. orf261a and orf322a were cotranscribed with cox1 and atp9, respectively. Additionally, orf114a was cotranscribed with atp8. Yeast two-hybrid assays showed that the ORF222a protein interacts with a B. oleracea ATP17 homolog (Bo7g114140) during F0-type ATP synthase assembly, reducing the quantity and activity of assembled F1F0-ATP synthase. Cytological sections showed that premature separation of the tapetum from the connective tissue and delayed tapetal programmed cell death (PCD) might be the immediate causes of CMS in C5-type CMS cabbage lines. Our results may help uncover the molecular mechanism of C5-type CMS in B. oleracea from the perspectives of the whole mitochondrial genome and cytology of anther development.

Comparative Genomic Analysis of Xanthomonas campestris pv. campestris Isolates BJSJQ20200612 and GSXT20191014 Provides Novel Insights Into Their Genetic Variability and Virulence.

Black rot is a disease that has a severe impact on cabbage yield and quality in China. Xanthomonas campestris pv. campestris (Xcc) is the causal agent of black rot of Brassicaceae crops. So far, the whole genomic sequences of more than 30 Xcc isolates have been sequenced; however, little information about genomic variability and virulence has been reported. In this study, 12 Xcc isolates were isolated from diseased cabbage leaves in seven Chinese provinces and two municipalities from July 2019 to November 2020. Pathogenicity analysis showed that isolate GSXT20191014 was more aggressive than BJSJQ20200612 and HRIW 3811 on cabbage inbred line 1371. Both BJSJQ20200612 and GSXT20191014 were sequenced and comparatively analyzed. The results showed that BJSJQ20200612 and GSXT20191014 have a single circular chromosome comprising 5,115,975 and 4,975,682 bp, respectively. Compared to the other six sequenced strains, 26 and 47 variable genomic regions were found in BJSJQ2020061 and GSXT20191014 genomic sequences, respectively. The variable genomic regions could be responsible for the genetic variation in Xcc strains and have led to the differences in type III secreted effector repertoires, virulence factors and secreted proteins between these two strains. Among the identified secreted proteins, two copies of peptidase S8/S53 were found in GSXT20191014-specific chromosomal segments. The common effectors xopR, xopH, avrBs1, and xopAH are found in most Xcc genomes, but they are absent in the GSXT20191014 genome. Variations in the composition of exopolysaccharides (EPS) and lipopolysaccharides (LPS) may aid GSXT20191014 isolate infections to evade recognition by the host immune system. Our results revealed a direct correlation between genomic variability and Xcc virulence. We also developed several markers for detecting BJSJQ20200612 and GSXT20191014 isolates and further tested the rest of our other 10 isolates. Finally, the isolated Xcc strains were classified into three genetic subgroups by specific molecular markers and multilocus sequence typing (MLST) approach. BJSJQ20200612 and GSXT20191014 isolates were also classified into two subgroups of Xcc according to the core-genome-based phylogenetic tree. This study extended our understanding of Xcc genomic features and provided the foundation to further characterize the mechanisms for Xcc virulence and a clue for black rot resistance breeding.

Comparative analysis of the complete mitochondrial genome sequences and anther development cytology between maintainer and Ogura-type cytoplasm male-sterile cabbage (B. oleracea Var. capitata).

BACKGROUND: Cytoplasmic male sterility (CMS) has been widely used for commercial F1 hybrid seeds production. CMS is primarily caused by chimeric genes in mitochondrial genomes. However, which specific stages of anther development in cabbage are affected by the chimeric genes remain unclear. RESULTS: In the present study, the complete mitochondrial genomes were sequenced and assembled for the maintainer and Ogura CMS cabbage lines. The genome size of the maintainer and Ogura CMS cabbage are 219,962 bp and 236,648 bp, respectively. There are 67 and 69 unknown function ORFs identified in the maintainer and Ogura CMS cabbage mitochondrial genomes, respectively. Four orfs, orf102a, orf122b, orf138a and orf154a were specifically identified in the Ogura CMS mitochondrial genome, which were likely generated by recombination with Ogura type radish during breeding process. Among them, ORF138a and ORF154a possessed a transmembrane structure, and orf138a was co-transcribed with the atp8 and trnfM genes. orf154a is partially homologous to the ATP synthase subunit 1 (atpA) gene. Both these genes were likely responsible for the CMS phenotype. In addition, cytological sections showed that the abnormal proliferation of tapetal cells might be the immediate cause of cytoplasmic male-sterility in Ogura CMS cabbage lines. RNA-seq results showed that orf138a and orf154a in Ogura CMS might influence transcript levels of genes in energy metabolic pathways. CONCLUSIONS: The presence of orf138a and orf154a lead to increased of ATPase activity and ATP content by affecting the transcript levels of genes in energy metabolic pathways, which could provide more energy for the abnormal proliferation of tapetal cells. Our data provides new insights into cytoplasmic male-sterility from whole mitochondrial genomes, cytology of anther development and transcriptome data.

Receptor-like kinases MDS1 and MDS2 promote SUMM2-mediated immunity.

Disruption of the MEKK1-MKK1/MKK2-MPK4 kinase cascade leads to activation of immunity mediated by the nucleotide-binding leucine-rich repeat (NLR) immune receptor SUMM2, which monitors the phosphorylation status of CRCK3. Here we report that two receptor-like kinases (RLKs), MDS1, and MDS2, function redundantly to promote SUMM2-mediated immunity. Activation of SUMM2-mediated immunity is dependent on MDS1, and to a less extent on MDS2. MDS1 associates with CRCK3 in planta and can phosphorylate CRCK3 in vitro, suggesting that it may target CRCK3 to positively regulate SUMM2-mediated signaling. Our finding highlights a new defense mechanism where RLKs promote NLR-mediated immunity.

A fungal effector targets a heat shock-dynamin protein complex to modulate mitochondrial dynamics and reduce plant immunity.

Mitochondria are essential for animal and plant immunity. Here, we report that the effector MoCDIP4 of the fungal pathogen Magnaporthe oryzae targets the mitochondria-associated OsDjA9-OsDRP1E protein complex to reduce rice immunity. The DnaJ protein OsDjA9 interacts with the dynamin-related protein OsDRP1E and promotes the degradation of OsDRP1E, which functions in mitochondrial fission. By contrast, MoCDIP4 binds OsDjA9 to compete with OsDRP1E, resulting in OsDRP1E accumulation. Knockout of OsDjA9 or overexpression of OsDRP1E or MoCDIP4 in transgenic rice results in shortened mitochondria and enhanced susceptibility to M. oryzae Overexpression of OsDjA9 or knockout of OsDRP1E in transgenic rice, in contrast, leads to elongated mitochondria and enhanced resistance to M. oryzae Our study therefore reveals a previously unidentified pathogen-infection strategy in which the pathogen delivers an effector into plant cells to target an HSP40-DRP complex; the targeting leads to the perturbation of mitochondrial dynamics, thereby inhibiting mitochondria-mediated plant immunity.

Identification and expression profiling analysis of NBS-LRR genes involved in Fusarium oxysporum f.sp. conglutinans resistance in cabbage.

As one of the most important resistance (R) gene families in plants, the NBS-LRR genes, encoding proteins with nucleotide-binding site (NBS) and leucine-rich repeat (LRR) domains, play significant roles in resisting pathogens. The published genomic data for cabbage (Brassica oleracea L.) provide valuable data to identify and characterize the genomic organization of cabbage NBS-LRR genes. Ultimately, we identified 105 TIR (N-terminal Toll/interleukin-1 receptor)-NBS-LRR (TNL) genes and 33 CC (coiled-coil)-NBS-LRR (CNL) genes. Further research indicated that 50.7% of the 138 NBS-LRR genes exist in 27 clusters and there are large differences among the gene structures and protein characteristics. Conserved motif and phylogenetic analysis showed that the structures of TNLs and CNLs were similar, with some differences. These NBS-LRRs are evolved under negative selection and mostly arose from whole-genome duplication events during evolution. Tissue-expression profiling of NBS-LRR genes revealed that 37.1% of the TNL genes are highly or specifically expressed in roots, especially the genes on chromosome 7 (76.5%). Digital gene expression and reverse transcription PCR analyses revealed the expression patterns of the NBS-LRR genes upon challenge by Fusarium oxysporum f.sp. conglutinans: nine genes were upregulated, and five were downregulated. The major resistance gene Foc1 probably works together with the other four genes in the same cluster to resist F. oxysporum infection.

Overexpression of lily HsfA3s in Arabidopsis confers increased thermotolerance and salt sensitivity via alterations in proline catabolism.

Although HsfA3 (heat-stress transcription factor A3) is well characterized in heat stress, its roles in other abiotic stresses are less clear. In this study, we isolated two homologous HsfA3 genes, LlHsfA3A and LlHsfA3B, from lily (Lilium longiflorum). Both genes were induced by heat stress, but not by salt stress. Overexpressing LlHsfA3A in Arabidopsis enhanced its basal and acquired thermotolerance, while overexpressing LlHsfA3B just enhanced its acquired thermotolerance. In both cases, overexpressing plants showed hypersensitivity to salt stress, and a lack of sucrose exacerbated this salt sensitivity. Using a transient assay, the opposite effects were observed in lily. Further analysis revealed that either LlHsfA3A or LlHsfA3B overexpression altered normal proline accumulation. During heat treatments, proline increased in wild-type Arabidopsis plants, but no such increase was detected in transgenic plants that showed better basal or acquired thermotolerance. Under salt stress, proline accumulation was decreased in Arabidopsis and lily with the overexpression of LlHsfA3A or LlHsfA3B. Proline catabolism was activated by overexpression, and both LlHsfA3A and LlHsfA3B affected proline oxidation via regulation of AtbZIP11, AtbZIP44, and AtbZIP53 to activate AtproDH1 and AtproDH2 in transgenic Arabidopsis. Taken together, our results suggested that overexpression of LlHsfA3A or LlHsfA3B caused opposite effects on heat and salt tolerance, which may implicate proline catabolism.

The DnaJ protein OsDjA6 negatively regulates rice innate immunity to the blast fungus Magnaporthe oryzae.

Rice blast, caused by Magnaporthe oryzae (synonym: Pyricularia oryzae), severely reduces rice production and grain quality. The molecular mechanism of rice resistance to M. oryzae is not fully understood. In this study, we identified a chaperone DnaJ protein, OsDjA6, which is involved in basal resistance to M. oryzae in rice. The OsDjA6 protein is distributed in the entire rice cell. The expression of OsDjA6 is significantly induced in rice after infection with a compatible isolate. Silencing of OsDjA6 in transgenic rice enhances resistance to M. oryzae and also results in an increased burst of reactive oxygen species after flg22 and chitin treatments. In addition, the expression levels of WRKY45, NPR1 and PR5 are increased in OsDjA6 RNAi plants, indicating that OsDjA6 may mediate resistance by affecting the salicylic acid pathway. Finally, we found that OsDjA6 interacts directly with the E3 ligase OsZFP1 in vitro and in vivo. These results suggest that the DnaJ protein OsDjA6 negatively regulates rice innate immunity, probably via the ubiquitination proteasome degradation pathway.

ADP-glucose pyrophosphorylase gene plays a key role in the quality of corm and yield of cormels in gladiolus.

Starch is the main storage compound in underground organs like corms. ADP-glucose pyrophosphorylase (AGPase) plays a key role in regulating starch biosynthesis in storage organs and is likely one of the most important determinant of sink strength. Here, we identify an AGPase gene (GhAGPS1) from gladiolus. The highest transcriptional levels of GhAGPS1 were observed in cormels and corms. Transformation of GhAGPS1 into Arabidopsis rescued the phenotype of aps1 mutant. Silencing GhAGPS1 in gladiolus corms by virus-induced gene silencing (VIGS) decreased the transcriptional levels of two genes and starch content. Transmission electron microscopy analyses of leaf and corm sections confirmed that starch biosynthesis was inhibited. Corm weight and cormel number reduced significantly in the silenced plants. Taken together, these results indicate that inhibiting the expression of AGPase gene could impair starch synthesis, which results in the lowered corm quality and cormel yield in gladiolus.

Plant TRAF Proteins Regulate NLR Immune Receptor Turnover.

In animals, Tumor necrosis factor receptor-associated factor (TRAF) proteins are molecular adaptors that regulate innate and adaptive immunity, development, and abiotic stress responses. Although gene families encoding TRAF domain-containing proteins exhibit enriched diversity in higher plants, their biological roles are poorly defined. Here, we report the identification of two redundant TRAF proteins, Mutant, snc1-enhancing 13 (MUSE13) and MUSE14, that contribute to the turnover of nucleotide-binding domain and leucine-rich repeat-containing (NLR) immune receptors SNC1 and RPS2. Loss of both MUSE13 and MUSE14 leads to enhanced pathogen resistance, NLR accumulation, and autoimmunity, while MUSE13 overexpression results in reduced NLR levels and activity. In planta, MUSE13 associates with SNC1, RPS2, and the E3 ubiquitin ligase SCF(CPR1). Taken together, we speculate that MUSE13 and MUSE14 associate with the SCF E3 ligase complex to form a plant-type TRAFasome, which modulates ubiquitination and subsequent degradation of NLR immune sensors to maintain their homeostasis.

The NPR1 homolog GhNPR1 plays an important role in the defense response of Gladiolus hybridus.

KEY MESSAGE: GhNPR1 shares similar functions as Arabidopsis NPR1 . Silencing of GhNPR1 in Gladiolus results in an enhanced susceptibility to Curvularia gladioli. We propose that GhNPR1 plays important roles in plant immunity. Gladiolus plants and corms are susceptible to various types of pathogens including fungi, bacteria and viruses. Understanding the innate defense mechanism in Gladiolus is a prerequisite for the development of new protection strategies. The non-expressor of pathogenesis-related gene 1 (NPR1) and bzip transcription factor TGA2 play a key role in regulating salicylic acid (SA)-mediated systemic acquired resistance (SAR). In this study, the homologous genes, GhNPR1 and GhTGA2, were isolated from Gladiolus and functionally characterized. Expression of GhNPR1 exhibited a 3.8-fold increase in Gladiolus leaves following salicylic acid treatment. A 1332 bp fragment of the GhNPR1 promoter from Gladiolus hybridus was identified. Inducibility of the GhNPR1 promoter by SA was demonstrated using transient expression assays in the leaves of Nicotiana benthamiana. The GhNPR1 protein is located in the nucleus and cytomembrane. GhNPR1 interacts with GhTGA2, as observed using the bimolecular fluorescence complementation system. Overexpression of GhNPR1 in an Arabidopsis npr1 mutant can restore its basal resistance to Pseudomonas syringae pv. tomato DC3000. Silencing of GhNPR1, using a tobacco rattle virus-based silencing vector, resulted in an enhanced susceptibility to Curvularia gladioli. In conclusion, these results suggest that GhNPR1 plays a pivotal role in the SA-dependent systemic acquired resistance in Gladiolus.

HSP90s are required for NLR immune receptor accumulation in Arabidopsis.

Heat shock proteins (HSPs) serve as molecular chaperones for diverse client proteins in many biological processes. In plant immunity, cytosolic HSP90s participate in the assembly, stability control and/or activation of immune receptor complexes. In this paper we report that in addition to the well-established positive roles that HSP90 isoforms play in plant immunity, they are also involved in the negative regulation of immune receptor accumulation. Point mutations in two HSP90 genes, HSP90.2 and HSP90.3, were identified from a forward genetic screen designed to isolate mutants with enhanced disease resistance. We found that specific mutations in HSP90.2 and HSP90.3 lead to heightened accumulation of immune receptors, including SNC1, RPS2 and RPS4. HSP90s may assist SGT1 in the formation of SCF E3 ubiquitin ligase complexes that target immune receptors for degradation. Such regulation is critical for maintaining appropriate levels of immune receptor proteins to avoid autoimmunity.

LlHSFA1, a novel heat stress transcription factor in lily (Lilium longiflorum), can interact with LlHSFA2 and enhance the thermotolerance of transgenic Arabidopsis thaliana.

KEY MESSAGE: A heat stress transcription factor LlHSFA1 in lily and its relationship with LlHSFA2 was investigated, and its function in enhancing thermotolerance was confirmed by analyzing transgenic Arabidopsis thaliana overexpressed LlHSFA1. A large family of heat stress transcription factors that are involved in the heat stress response in plants can induce the expression of multiple genes related to thermotolerance including heat-shock proteins. In this study, a novel class A1 HSF named LlHSFA1 was isolated from leaves of lily (Lilium longiflorum cv. 'White Heaven') using the rapid amplification of cDNA ends technique. Analysis of the deduced amino acid sequence and construction of a phylogenetic tree showed that LlHSFA1 contained five critical domains and motifs and belonged to the A1 family of HSFs. Following the heat treatment of lily leaves, transcription of LlHSFA1 was induced to a varying extent, related to the time of measurement. The induced expression peak of LlHSFA1 occurred prior to that of LlHSFA2, during the early phase of heat stress. Following transient expression of LlHSFA1 in Nicotiana benthamiana, LlHSFA1 was found to be localized in both the nucleus and the cytoplasm. Analysis using bimolecular fluorescence complementation and a yeast two-hybrid assay demonstrated that LlHSFA1 could interact with LlHSFA2. Use of a yeast one-hybrid assay confirmed that LlHSFA1 had transcriptional activation activity. In transgenic Arabidopsis lines overexpressing LlHSFA1 under unstressed conditions, the expression of some putative target genes was up-regulated, in comparison with expression in wild-type plants, and furthermore, the thermotolerance of the transgenic lines was enhanced. Overall, LlHSFA1 was demonstrated to play an important role in the heat stress response of lily and to be a novel candidate gene for application in lily breeding, using genetic modification approaches.

Virus-induced gene silencing for comparative functional studies in Gladiolus hybridus.

Functional analysis of genes in gladiolus has previously been impractical due to the lack of an efficient stable genetic transformation method. However, virus-induced gene silencing (VIGS) is effective in some plants which are difficult to transform through other methods. Although the Tobacco rattle virus (TRV)-based VIGS system has been developed and used for verifying gene functions in diverse plants, an appropriate TRV-VIGS approach for gladiolus has not been established yet. In this report we describe the first use of the TRV-VIGS system for gene silencing in gladiolus. Vacuum infiltration of cormels and young plants with the GhPDS-VIGS vector effectively down-regulated the PHYTOENE DESATURASE ortholog GhPDS gene and also resulted in various degrees of photobleaching in Gladiolus hybridus. The reduction in GhPDS expression was tested after TRV-based vector infection using real-time RT-PCR. In addition, the progress of TRV infection was detected by fluorescence visualization using a pTRV2: CP-GFP vector. In conclusion, the TRV-mediated VIGS described here will be an effective gene function analysis mechanism in gladiolus.