Myosin XI-mediated BIK1 recruitment to nanodomains facilitates FLS2-BIK1 complex formation during innate immunity in Arabidopsis.

Plants rely on immune receptor complexes at the cell surface to perceive microbial molecules and transduce these signals into the cell to regulate immunity. Various immune receptors and associated proteins are often dynamically distributed in specific nanodomains on the plasma membrane (PM). However, the exact molecular mechanism and functional relevance of this nanodomain targeting in plant immunity regulation remain largely unknown. By utilizing high spatiotemporal resolution imaging and single-particle tracking analysis, we show that myosin XIK interacts with remorin to recruit and stabilize PM-associated kinase BOTRYTIS-INDUCED KINASE 1 (BIK1) within immune receptor FLAGELLIN SENSING 2 (FLS2)-containing nanodomains. This recruitment facilitates FLS2/BIK1 complex formation, leading to the full activation of BIK1-dependent defense responses upon ligand perception. Collectively, our findings provide compelling evidence that myosin XI functions as a molecular scaffold to enable a spatially confined complex assembly within nanodomains. This ensures the presence of a sufficient quantity of preformed immune receptor complex for efficient signaling transduction from the cell surface.

Analysis of Actin Array Rearrangement During the Plant Response to Bacterial Stimuli.

Plants are constantly exposed to various environmental stresses, among which, microbial pathogens are one of the major threats. Studies have shown that the host actin cytoskeleton undergoes active rearrangement during the plant-microbe interaction. This actin remodeling is required for plant resistance to bacterial infection. In this chapter, we introduce a protocol routinely used in our laboratory to investigate actin dynamics in response to bacterial cues. We describe the bacterial inoculation methods, plant sample preparation, and imaging techniques used to monitor actin responses in different Arabidopsis cell types including epidermal cells from light-grown leaves and dark-grown hypocotyls, as well as guard cells. We further introduce a high-throughput image analysis method for quantifying cytoskeletal changes. This protocol has allowed us to dissect the host cell contribution to actin remodeling and identify actin-binding proteins as stimulus-response regulators of the cytoskeleton.

MPK3- and MPK6-mediated VLN3 phosphorylation regulates actin dynamics during stomatal immunity in Arabidopsis.

Upon perception of pathogens, plants can rapidly close their stomata to restrict pathogen entry into internal tissue, leading to stomatal immunity as one aspect of innate immune responses. The actin cytoskeleton is required for plant defense against microbial invaders. However, the precise functions of host actin during plant immunity remain largely unknown. Here, we report that Arabidopsis villin3 (VLN3) is critical for plant resistance to bacteria by regulating stomatal immunity. Our in vitro and in vivo phosphorylation assays show that VLN3 is a physiological substrate of two pathogen-responsive mitogen-activated protein kinases, MPK3/6. Quantitative analyses of actin dynamics and genetic studies reveal that VLN3 phosphorylation by MPK3/6 modulates actin remodeling to activate stomatal defense in Arabidopsis.

The landscape of transposable elements and satellite DNAs in the genome of a dioecious plant spinach (Spinacia oleracea L.).

BACKGROUND: Repetitive sequences, including transposable elements (TEs) and satellite DNAs, occupy a considerable portion of plant genomes. Analysis of the repeat fraction benefits the understanding of genome structure and evolution. Spinach (Spinacia oleracea L.), an important vegetable crop, is also a model dioecious plant species for studying sex determination and sex chromosome evolution. However, the repetitive sequences of the spinach genome have not been fully investigated. RESULTS: We extensively analyzed the repetitive components of draft spinach genome, especially TEs and satellites, by different strategies. A total of 16,002 full-length TEs were identified. Among the most abundant long terminal repeat (LTR) retrotransposons (REs), Copia elements were overrepresented compared with Gypsy ones. Angela was the most dominating Copia lineage; Ogre/Tat was the most abundant Gypsy lineage. The mean insertion age of LTR-REs was 1.42 million years; approximately 83.7% of these elements were retrotransposed during the last two million years. RepeatMasker totally masked about 64.05% of the spinach genome, with LTR-REs, non-LTR-REs, and DNA transposons occupying 49.2, 2.4, and 5.6%, respectively. Fluorescence in situ hybridization (FISH) analysis showed that most LTR-REs dispersed all over the chromosomes, by contrast, elements of CRM lineage were distributed at the centromeric region of all chromosomes. In addition, Ogre/Tat lineage mainly accumulated on sex chromosomes, and satellites Spsat2 and Spsat3 were exclusively located at the telomeric region of the short arm of sex chromosomes. CONCLUSIONS: We reliably annotated the TE fraction of the draft genome of spinach. FISH analysis indicates that Ogre/Tat lineage and the sex chromosome-specific satellites DNAs might participate in sex chromosome formation and evolution. Based on FISH signals of microsatellites, together with 45S rDNA, a fine karyotype of spinach was established. This study improves our knowledge of repetitive sequence organization in spinach genome and aids in accurate spinach karyotype construction.

Genome-wide characterization of microsatellites and genetic diversity assessment of spinach in the Chinese germplasm collection.

Spinach is a nutritional leafy green vegetable, and it also serves as a model species for studying sex chromosome evolution. Genetic marker development and genome structure analysis are important in breeding practice and theoretical evolution studies of spinach. In this study, the frequency and distribution of different microsatellites in the recently released draft spinach genome were characterized. A total of 261,002 perfect microsatellites were identified (estimated frequency: ~262.1 loci/Mbp). The most abundant microsatellites were tetranucleotide and trinucleotide, accounting for 33.2% and 27.7% of the total number of microsatellites, respectively. A total of 105 primer pairs were designed and screened, and 34 were polymorphic among the detected spinach cultivars. Combined with seven primer sets developed previously, 41 primer pairs were used to investigate genetic diversity among 43 spinach cultivars in China. The average polymorphism information content value of the 41 markers was 0.43, representing an intermediate level. The spinach cultivars had a low genetic diversity, and no detectable common factors were shared by each group in the UPGMA dendrogram. This study's findings facilitate further investigations on the organization of the microsatellites in spinach genome and provide clues for future breeding applications of spinach in China.

Chromosome Evolution in Connection with Repetitive Sequences and Epigenetics in Plants.

Chromosome evolution is a fundamental aspect of evolutionary biology. The evolution of chromosome size, structure and shape, number, and the change in DNA composition suggest the high plasticity of nuclear genomes at the chromosomal level. Repetitive DNA sequences, which represent a conspicuous fraction of every eukaryotic genome, particularly in plants, are found to be tightly linked with plant chromosome evolution. Different classes of repetitive sequences have distinct distribution patterns on the chromosomes. Mounting evidence shows that repetitive sequences may play multiple generative roles in shaping the chromosome karyotypes in plants. Furthermore, recent development in our understanding of the repetitive sequences and plant chromosome evolution has elucidated the involvement of a spectrum of epigenetic modification. In this review, we focused on the recent evidence relating to the distribution pattern of repetitive sequences in plant chromosomes and highlighted their potential relevance to chromosome evolution in plants. We also discussed the possible connections between evolution and epigenetic alterations in chromosome structure and repatterning, such as heterochromatin formation, centromere function, and epigenetic-associated transposable element inactivation.

Amino acid substitutions in the neuraminidase protein of an H9N2 avian influenza virus affect its airborne transmission in chickens.

Cases of H9N2 avian influenza virus (AIV) in poultry are increasing throughout many Eurasian countries, and co-infections with other pathogens have resulted in high morbidity and mortality in poultry. Few studies have investigated the genetic factors of virus airborne transmission which determine the scope of this epidemic. In this study, we used specific-pathogen-free chickens housed in isolators to investigate the airborne transmissibility of five recombinant H9N2 AIV rescued by reverse genetic technology. The results show that airborne transmission of A/Chicken/Shandong/01/2008 (SD01) virus was related to the neuraminidase (NA) gene, and four amino acid mutations (D368E, S370L, E313K and G381D) within the head region of the SD01 NA, reduced virus replication in the respiratory tract of chickens, reduced virus NA activity, and resulted in a loss of airborne transmission ability in chickens. Similarly, reverse mutations of these four amino acids in the NA protein of r01/NASS virus, conferred an airborne transmission ability to the recombinant virus. We conclude that these four NA residues may be significant genetic markers for evaluating potential disease outbreak of H9N2 AIV, and propose that immediate attention should be paid to the airborne transmission of this virus.