Silenced Transformants Research Articles

PcLRR-RK3, an LRR receptor kinase is required for growth and in-planta infection processes in Phytophthora capsici

ABSTRACT Receptor protein kinases (RPKs) critically provide the basic infrastructure to sense, perceive, and conduct the signalling events at the cell surface of organisms. The importance of LRR-RLKs has been well studied in plants, but much less information has been reported in oomycetes. In this work, we have silenced the PcLRR-RK3 and characterised its functional importance in Phytophthora capsici. PcLRR-RK3 was predicted to encode signal peptides, leucine-rich repeats, transmembrane, and kinase domains. PcLRR-RK3-silenced transformants showed impaired colony growth, decreased deformed sporangia, and reduced zoospores count. The mycelium of silenced transformants did not penetrate within the host tissues and showed defects in the pathogenicity of P. capsici. Interestingly, gene silencing also weakens the ability of zoospores germination and penetration into host tissues and fails to produce necrotic lesions. Furthermore, PcLRR-RK3 localisation was found to be the plasma membrane of the cell. Altogether, our results revealed that PcLRR-RK3 was required for the regulation of vegetative growth, zoospores penetration, and establishment into host leaf tissues.

Engineering T cell receptor fusion proteins using nonviral CRISPR/Cas9 genome editing for cancer immunotherapy.

Manufacture of chimeric antigen receptor (CAR)-T cells usually involves the use of viral delivery systems to achieve high transgene expression. However, it can be costly and may result in random integration of the CAR into the genome, creating several disadvantages including variation in transgene expression, functional gene silencing and potential oncogenic transformation. Here, we optimized the method of nonviral, CRISPR/Cas9 genome editing using large donor DNA delivery, knocked-in an anti-tumor single chain variable fragment (scFv) into the N-terminus of CD3ε and efficiently generated fusion protein (FP) T cells. These cells displayed FP integration within the TCR/CD3 complex, lower variability in gene expression compared to CAR-T cells and good cell expansion after transfection. CD3ε FP T cells were predominantly CD8+ effector memory T cells, and exhibited anti-tumor activity in vitro and in vivo. Dual targeting FP T cells were also generated through the incorporation of scFvs into other CD3 subunits and CD28. Compared to viral-based methods, this method serves as an alternative and versatile way of generating T cells with tumor-targeting receptors for cancer immunotherapy.

Identification of genes for seed isoflavones based on bulk segregant analysis sequencing in soybean natural population.

We identified four hub genes for isoflavone biosynthesis based on BSA-seq and WGCNA methods and validated that GmIE3-1 positively contribute to isoflavone accumulation in soybean. Soybean isoflavones are secondary metabolites of great interest owing to their beneficial impact on human health. Herein, we profiled the seed isoflavone content by HPLC in 1551 soybean accessions grown in two locations for two years and constructed two extreme pools with high (4065.1µgg-1) and low (1427.23µgg-1) isoflavone contents to identify candidate genes involved in isoflavone biosynthesis pathways using bulk segregant analysis sequencing (BSA-seq) approach. The results showed that the average sequencing depths were 50.3× and 65.7× in high and low pools, respectively. A total of 23,626 polymorphic SNPs and 5299 InDels were detected between both pools and 1492 genes with different variations were identified. Based on differential genes in BSA-seq and weighted gene co-expression network analysis (WGCNA), four hub genes, Glyma.06G290400 (designated as GmIE3-1), Glyma.01G239200, Glyma.01G241500, Glyma.13G256100 were identified, encoding E3 ubiquitin-protein ligase, arm repeat protein interacting with ABF2, zinc metallopeptidase EGY3, and dynamin-related protein 3A, respectively. The allelic variation in GmIE3-1 showed a significant influence on isoflavone accumulation. The virus-induced gene silencing (VIGS) and RNAi hairy root transformation of GmIE3-1 revealed partial suppression of this gene could cause a significant decrease (P < 0.0001) of total isoflavone content, suggesting GmIE3-1 is a positive regulator for isoflavones. The present study demonstrated that the BSA-seq approach combined with WGCNA, VIGS and hairy root transformation can efficiently identify isoflavone candidate genes in soybean natural population.

DNA Methylation: From Cancer Biology to Clinical Perspectives.

DNA methylation plays an important role in the silence of tissue-specific genes to prevent them from being expressed in the wrong tissue. Aberrant DNA methylation (genome-wide hypomethylation and site-specific hypermethylation) are observed in many types of cancer. DNA methylation patterns are established and maintained through the combined actions of methyltransferase and demethylase, such as DNA methyltransferase (DNMT)-1, DNMT-3, and ten-eleven translocation (TET) family enzymes. It is well known that the process of tumor evolution is complicated with different hallmarks. Early findings put forward the model that focal hypermethylation of tumor suppressor genes (TSG) could straightly trigger transcriptional silencing and malignant transformation, whereas varying levels of DNA methylation also occur at other sites and can differently regulate gene expression and biological processes. The interplay of tumor and immune cells in the tumor microenvironment is complex. Understanding the role of DNA methylation in cancer immunity is critical to better navigate epigenetic agents. Furthermore, a greater understanding of the interaction of DNA methylation with tumor metabolic reprogramming would create a bright avenue for pharmacologic managements of malignancies. In this review, we will describe the molecular mechanisms of DNA methylation abnormalities in cancer biology, introduce the roles of DNA methylation patterns on cancer-immunity cycle and metabolic reprogramming, summarize modulators that are used in targeting DNA remodeling, and highlight the importance of combining epigenome-targeting drugs with other cancer therapies.

Hannah’s Suffering: The Power of Voice

Hannah’s story in the Old Testament has been written about considerably by Jewish feminists, womanist theologians, and other biblical scholars. This paper strives to build upon these works in asking the reader to consider Hannah’s story from a liberatory theological theory of suffering by Sölle, as well as a postmodern and non-religious lens as discussed by Sandoval’s Theory of Oppositional Consciousness in Methodology of the Oppressed and Lorde’s “Transformation of Silence into Language and Action”. This paper asks if this narrative can serve as an example of taking back one’s power by confronting a complex system of power and oppression for Black women. Intercessory prayer aptly defines the personal as political, especially with the multiple minoritized identities of Hannah. I argue that Hannah’s story can serve as a complex narrative of differential consciousness and the reclamation of one’s own power, by using her voice. Her audacity to correct a prophet, fight for her valid desire of motherhood, and determine her own happiness is evidence of an empowerment ethic that is necessary for minoritized women in a post-modern era and political climate where the erasure of all forms of difference and consciousness is the priority.

A Transformation of Shame and Silence

ABSTRACT Societal hauntings overlap with, and yet are distinct from, individual traumas. They are nested systems with interrelated dynamics. Weaving insights from clinical interactions, an incident in a professional space, and her personal experiences, the author explores conceptual expansions that move clinical and personal work beyond the realm of trauma-based language of victim and perpetrator into the realm of implication. Examples of how societal hauntings inform the shape of individual and familial trauma are explored as the author acknowledges the interpenetrating and clashing experiences of injustice, trauma, and privilege. The article concludes with a reminder of how psychoanalysis can be a crucible in which these contradictory experiences can be welcomed and transformed if we are willing to move through our own shame and beyond our individual and collective silence.

In Vivo Functional Verification of Four Related Genes Involved in the 1-Deoxynojirimycin Biosynthetic Pathway in Mulberry Leaves.

The alkaloid 1-deoxynojirimycin (DNJ) is one of the major bioactive compounds in mulberry leaves (Morus alba L.). Previously, we discovered four key genes involved in the pathway from lysine to piperidine in the biosynthesis of DNJ in mulberry leaves, MaLDC (MG727866), MaCAO (MH205733), MaSDR1 (MT989445), and MaSDR2 (MT989446), which encoded lysine decarboxylase, copper amine oxidase, and short-chain dehydrogenase/reductase 1 and 2, respectively. However, the in vivo functions of these four genes have not been verified yet. Here, these four genes were successfully cloned and used for the establishment of C58C1 Agrobacterium rhizogenes mediated overexpression genetic transformation systems and GV3101 Agrobacterium-mediated virus-induced gene silencing transformation systems in order to verify the influence of these four genes on the biosynthetic content of DNJ in mulberry leaves. The results showed that the content of DNJ increased after the four genes were overexpressed. When these four genes were silenced, the gene expression was blocked, which affected the biosynthesis of DNJ, and the DNJ content decreased. The above results indicated that these four genes participated in DNJ biosynthesis. This study provided a foundation for further elucidating the regulatory mechanisms of DNJ biosynthesis in mulberry leaves.

Editor’s Introduction

Editorial| March 01 2021 Editor’s Introduction Devika Chawla Devika Chawla Devika Chawla is Professor in the School of Communication Studies at Ohio University. Correspondence to: Devika Chawla, School of Communication Studies, 431 Schoonover Center, Ohio University, Athens, OH 45701, USA. Email: chawla@ohio.edu. Search for other works by this author on: This Site PubMed Google Scholar Departures in Critical Qualitative Research (2021) 10 (1): 1–3. https://doi.org/10.1525/dcqr.2021.10.1.1 Views Icon Views Article contents Figures & tables Video Audio Supplementary Data Peer Review Share Icon Share Twitter LinkedIn Tools Icon Tools Get Permissions Cite Icon Cite Search Site Citation Devika Chawla; Editor’s Introduction. Departures in Critical Qualitative Research 1 March 2021; 10 (1): 1–3. doi: https://doi.org/10.1525/dcqr.2021.10.1.1 Download citation file: Ris (Zotero) Reference Manager EasyBib Bookends Mendeley Papers EndNote RefWorks BibTex toolbar search Search Dropdown Menu toolbar search search input Search input auto suggest filter your search All ContentDepartures in Critical Qualitative Research Search By the time this Introduction sees publication, it will be March 2021, and hopefully the United States will have inaugurated a new president and the first female, Black, and South Asian vice president. I am writing on 8 November 2020, one day after one of the most fraught presidential races in American history—what was fundamentally a contest between the forces of inclusion and exclusion in contemporary America. It is indeed prescient that the essays in this issue of Departures in Critical Qualitative Research (10.1) navigate the themes of inclusion/exclusion, native/Other, insiderness/outsiderness, survival, stigma, trauma, and colonial histories. How do Black women survive, thrive, and emerge as leaders in predominantly white and systemically racist institutions such as politics and academics? In “The Transformation of Silence into Language and Action,” Audre Lorde writes:Black women have on one hand always been highly visible, and so, on the other hand, have been rendered... You do not currently have access to this content.

The Trichoderma harzianum Kelch Protein ThKEL1 Plays a Key Role in Root Colonization and the Induction of Systemic Defense in Brassicaceae Plants.

The fungal genus Trichoderma includes strains with biocontrol and/or biostimulant potential and is recognized as a source of genes with biotechnological value. In a previous study the Kelch domain protein, encoded by the Thkel1 gene of Trichoderma harzianum T34, was found to confer tolerance to salt stress when expressed in plants of Arabidopsis thaliana. In the present work, we have overexpressed Thkel1 in rapeseed plants in order to generate an additional biotechnological tool for analyzing the role of this gene in Trichoderma-plant interactions. The overexpression of this gene in Brassicaceae plants improves responses to pathogens through the induction of systemic defenses mediated by jasmonic acid, facilitates root colonization by modulating the myrosinase activity, and, as a result, increases plant productivity. These effects were also observed in Thkel1 overexpressing plants subjected to abiotic stress conditions. Additionally, the differences detected in root colonization levels by T. harzianum wild type and Thkel1 silenced transformants between Arabidopsis or rapeseed and tomato plants indicate that ThKEL1 interacts in different ways in Brassicaceae and non-Brassicaceae plants.

Transient Expression Assay in Strawberry Fruits.

Strawberry, including the woodland strawberry Fragaria vesca (2x) and the cultivated strawberry (Fragaria × ananassa, 8x), has emerged as a model system for studying fruit development and ripening. Transient expression provides a quick assay for gene functions or gene interactions. In strawberry, virus-induced gene silencing (VIGS) and Agrobacterium tumefaciens-mediated transformation in fruit have been widely used as the transient expression approaches. Unlike VIGS, the latter one can be utilized not only for gene knock-down, but also for overexpression and knock-out. Here, we show the procedures of transiently expressing the 35S::FveMYB10 construct into fruit of the white-fruited F. vesca accession Yellow Wonder. As a master regulator of anthocyanin production, overexpressing FveMYB10 will cause fruit coloration, which was observed at one week post infiltration. We also exhibit the previous results of knocking down Reduced Anthocyanin in Petioles (RAP), encoding an anthocyanin transporter, by RNAi in fruit of the strawberry cultivar 'Sweet Charlie'. Overall, Agrobacterium-mediated transient transformation in strawberry fruit is a quick and versatile approach for studying gene functions in fruit ripening.

Agrobacterium-mediated and electroporation-mediated transformation of Chlamydomonas reinhardtii: a comparative study

BackgroundChlamydomonas reinhardtii is an unicellular green alga used for functional genomics studies and heterologous protein expression. A major hindrance in these studies is the low level and instability of expression of nuclear transgenes, due to their rearrangement and/or silencing over time.ResultsWe constructed dedicated vectors for Agrobacterium-mediated transformation carrying, within the T-DNA borders, the Paromomycin (Paro) selectable marker and an expression cassette containing the Luciferase (Luc) reporter gene. These vectors and newly developed co-cultivation methods were used to compare the efficiency, stability and insertion sites of Agrobacterium- versus electroporation-mediated transformation. The influence of different transformation methods, of the cell wall, of the virulence of different Agrobacterium strains, and of transgene orientation with respect to T-DNA borders were assessed. False positive transformants were more frequent in Agrobacterium-mediated transformation compared to electroporation, compensating for the slightly lower proportion of silenced transformants observed in Agrobacterium-mediated transformation than in electroporation. The proportion of silenced transformants remained stable after 20 cycles of subculture in selective medium. Next generation sequencing confirmed the nuclear insertion points, which occurred in exons or untraslated regions (UTRs) for 10 out of 10 Agrobacterium-mediated and 9 out of 13 of electroporation-mediated insertions. Electroporation also resulted in higher numbers of insertions at multiple loci.ConclusionsDue to its labor-intensive nature, Agrobacterium transformation of Chlamydomonas does not present significant advantages over electroporation, with the possible exception of its use in insertional mutagenesis, due to the higher proportion of within-gene, single-locus insertions. Our data indirectly support the hypothesis that rearrangement of transforming DNA occurs in the Chlamydomonas cell, rather than in the extracellular space as previously proposed.

SsSm1, a Cerato-platanin family protein, is involved in the hyphal development and pathogenic process of Sclerotinia sclerotiorum

The filamentous fungus Sclerotinia sclerotiorum is an important plant pathogen with a worldwide distribution. It can infect a wide variety of plants, causing serious disease in many types of crops, such as rapeseed, sunflower and soybean. Sclerotinia stem rot caused by this fungus affects main crops and has led to great economic loss. Elicitors are a group of compounds that inspire the host plant to produce an immune response against invading pathogens. This study describes a protein that has high homology with the Trichoderma elicitor Sm1 and was found in the genome of S. sclerotiorum. We named this protein SsSm1. To determine whether this protein has an elicitor function like its homology protein, we constructed a heterologous expression vector for SsSm1 and expressed it in Escherichia coli. The protein of heterologous expression led to the formation of lesions in tobacco that closely resemble hypersensitive response lesions. Transient expression of the encoding gene of SsSm1 in tobacco leaves also caused hypersensitive response. Then, RNA silencing was used to identify the function of SsSm1. The hyphal growth and pathogenicity of silenced transformants were shown to be obviously lagging and branched abnormally. Transformants produced less infection cushions and deformed sclerotiorum. In addition, SsSm1 silencing caused weak tolerance to NaCl, sorbitol and SDS, and the sensitivity of mutants to carbendazim was also significantly decreased. Based on the above results, we speculate that this protein may be related to the development of hyphae, infection cushions and sclerotiorum, but the specific molecular mechanism needs to be studied further.

Silencing of DND1 in potato and tomato impedes conidial germination, attachment and hyphal growth of Botrytis cinerea

BackgroundBotrytis cinerea, a necrotrophic pathogenic fungus, attacks many crops including potato and tomato. Major genes for complete resistance to B. cinerea are not known in plants, but a few quantitative trait loci have been described in tomato. Loss of function of particular susceptibility (S) genes appears to provide a new source of resistance to B. cinerea in Arabidopsis.ResultsIn this study, orthologs of Arabidopsis S genes (DND1, DMR6, DMR1 and PMR4) were silenced by RNAi in potato and tomato (only for DND1). DND1 well-silenced potato and tomato plants showed significantly reduced diameters of B. cinerea lesions as compared to control plants, at all-time points analysed. Reduced lesion diameter was also observed on leaves of DMR6 silenced potato plants but only at 3 days post inoculation (dpi). The DMR1 and PMR4 silenced potato transformants were as susceptible as the control cv Desiree. Microscopic analysis was performed to observe B. cinerea infection progress in DND1 well-silenced potato and tomato leaves. A significantly lower number of B. cinerea conidia remained attached to the leaf surface of DND1 well-silenced potato and tomato plants and the hyphal growth of germlings was hampered.ConclusionsThis is the first report of a cytological investigation of Botrytis development on DND1-silenced crop plants. Silencing of DND1 led to reduced susceptibility to Botrytis, which was associated with impediment of conidial germination and attachment as well as hyphal growth. Our results provide new insights regarding the use of S genes in resistance breeding.

RNA silencing of PEX6 gene causes decrease in pigmentation, sporulation and pathogenicity of Fusarium oxysporum

Peroxisomes are single membrane‐bound organelles that play a pivotal role in various developmental processes in all eukaryotic cells. This study targeted the PEX6 gene, which encodes for peroxisomal biogenesis factor 6, by RNA interference (RNAi) in Fusarium oxysporum f. sp. lycopersici. Fusarium oxysporum is a soilborne filamentous, hemibiotrophic fungus that invades tomato roots and colonizes the xylem vessels, thereby causing complete wilting of infected tomato plants. The expression of FoPEX6 in F. oxysporum was found to be higher during early stages of growth and development. The FoPEX6 gene was isolated and a hairpin RNAi construct was prepared and introduced into F. oxysporum 4471 through glass‐bead transformation. The fungal transformation status, i.e. integration, expression and presence of the intended small interfering RNAs (siRNAs), was confirmed by PCR, qPCR and stem‐loop PCR, respectively. The silenced fungal transformants exhibited reduced pigmentation and a significant reduction in sporulation as compared to the wild type. They also showed dramatic reduction in pathogenicity (virulence) on tomato, based on root infection and fruit invasion assays. These results suggest that PEX6 has a central role in pigmentation, sporulation and pathogenicity in F. oxysporum.

The Loricrin-Like Protein (LLP) of Phytophthora infestans Is Required for Oospore Formation and Plant Infection.

Loricrin-like protein (LLP) is characterized by a high content of glycine residues and is a major component of plant cell wall. Here, we identified a Phytophthora infestans ortholog of plant LLP, named PiLLP. In P. infestans, PiLLP is strongly expressed in asexual and sexual developmental stages, including in sporangia, zoospores and germinating cysts, and during oospore formation, as well as in the early stages of infection and during hydrogen peroxide stress. Compared with the wild type, the PiLLP-silenced transformants were defective in oospore formation, had slower colony expansion rates, produced less sporangia with lower germination and zoospore-release rates, and were more sensitive to hydrogen peroxide. Moreover, Nile red staining, and PiLLP-red fluorescent protein fusions indicated that PiLLP is involved in oogonia formation. The silenced transformants also had severely diminished virulence levels that could be partially restored with diphenyleneiodium treatments. The analysis of catalase activity showed a decrease of catalase activity in silenced transformants. Thus, PiLLP is important for sexual and asexual reproduction, and is required for oxidative stress tolerance and plant infection.

Laccase Gene Sh-lac Is Involved in the Growth and Melanin Biosynthesis of Scleromitrula shiraiana.

Scleromitrula shiraiana causes the popcorn disease in mulberry trees resulting in severe economic losses. Previous studies have shown that melanin may play a vital role in establishing the pathogenicity of fungi. In the present study, we identified the melanin produced in S. shiraiana belongs to DHN melanin by gas chromatography-mass spectrometry, and cloned the laccase Sh-lac, a potential DHN melanin biosynthesis gene from S. shiraiana. We obtained two stable Sh-lac silenced transformants using RNAi, ilac-4 and 8 to elucidate the DHN melanin biosynthetic pathway in S. shiraiana. The melanin production of ilac-4 and ilac-8 was significantly reduced, and their vegetative growth was also suppressed. Results such as these led to a proposal that Sh-lac played a key role in DHN melanin formation in S. shiraiana and may function differentially with other melanin biosynthetic genes. The inhibition of melanin was accompanied by the decrease of oxalic acid and the adhesion of hyphae was impaired. Our results indicated that laccase was an important enzyme in the synthesis of melanin and might play a critical role in the pathogenicity of S. shiraiana.

The importance of chorismate mutase in the biocontrol potential of Trichoderma parareesei.

Species of Trichoderma exert direct biocontrol activity against soil-borne plant pathogens due to their ability to compete for nutrients and to inhibit or kill their targets through the production of antibiotics and/or hydrolytic enzymes. In addition to these abilities, Trichoderma spp. have beneficial effects for plants, including the stimulation of defenses and the promotion of growth. Here we study the role in biocontrol of the T. parareesei Tparo7 gene, encoding a chorismate mutase (CM), a shikimate pathway branch point leading to the production of aromatic amino acids, which are not only essential components of protein synthesis but also the precursors of a wide range of secondary metabolites. We isolated T. parareesei transformants with the Tparo7 gene silenced. Compared with the wild-type, decreased levels of Tparo7 expression in the silenced transformants were accompanied by reduced CM activity, lower growth rates on different culture media, and reduced mycoparasitic behavior against the phytopathogenic fungi Rhizoctonia solani, Fusarium oxysporum and Botrytis cinerea in dual cultures. By contrast, higher amounts of the aromatic metabolites tyrosol, 2-phenylethanol and salicylic acid were detected in supernatants from the silenced transformants, which were able to inhibit the growth of F. oxysporum and B. cinerea. In in vitro plant assays, Tparo7-silenced transformants also showed a reduced capacity to colonize tomato roots. The effect of Tparo7-silencing on tomato plant responses was examined in greenhouse assays. The growth of plants colonized by the silenced transformants was reduced and the plants exhibited an increased susceptibility to B. cinerea in comparison with the responses observed for control plants. In addition, the plants turned yellowish and were defective in jasmonic acid- and ethylene-regulated signaling pathways which was seen by expression analysis of lipoxygenase 1 (LOX1), ethylene-insensitive protein 2 (EIN2) and pathogenesis-related protein 1 (PR-1) genes.

Phytophthora capsici homologue of the cell cycle regulator SDA1 is required for sporangial morphology, mycelial growth and plant infection.

SDA1 encodes a highly conserved protein that is widely distributed in eukaryotic organisms. SDA1 is essential for cell cycle progression and organization of the actin cytoskeleton in yeasts and humans. In this study, we identified a Phytophthora capsici orthologue of yeast SDA1, named PcSDA1. In P. capsici, PcSDA1 is strongly expressed in three asexual developmental states (mycelium, sporangia and germinating cysts), as well as late in infection. Silencing or overexpression of PcSDA1 in P. capsici transformants affected the growth of hyphae and sporangiophores, sporangial development, cyst germination and zoospore release. Phalloidin staining confirmed that PcSDA1 is required for organization of the actin cytoskeleton. Moreover, 4',6-diamidino-2-phenylindole (DAPI) staining and PcSDA1-green fluorescent protein (GFP) fusions revealed that PcSDA1 is involved in the regulation of nuclear distribution in hyphae and sporangia. Both silenced and overexpression transformants showed severely diminished virulence. Thus, our results suggest that PcSDA1 plays a similar role in the regulation of the actin cytoskeleton and nuclear division in this filamentous organism as in non-filamentous yeasts and human cells.

A KNOTTED1-LIKE HOMEOBOX protein regulates abscission in tomato by modulating the auxin pathway.

A gene encoding a KNOTTED1-LIKE HOMEOBOX PROTEIN1 (KD1) is highly expressed in both leaf and flower abscission zones. Reducing the abundance of transcripts of this gene in tomato (Solanum lycopersicum) by both virus-induced gene silencing and stable transformation with a silencing construct driven by an abscission-specific promoter resulted in a striking retardation of pedicel and petiole abscission. In contrast, Petroselinum, a semidominant KD1 mutant, showed accelerated pedicel and petiole abscission. Complementary DNA microarray and quantitative reverse transcription-polymerase chain reaction analysis indicated that regulation of abscission by KD1 was associated with changed abundance of genes related to auxin transporters and signaling components. Measurement of auxin content and activity of a DR5::β-glucuronidase auxin reporter assay showed that changes in KD1 expression modulated the auxin concentration and response gradient in the abscission zone.

Characterization of necrosis-inducing NLP proteins in Phytophthora capsici.

BackgroundEffector proteins function not only as toxins to induce plant cell death, but also enable pathogens to suppress or evade plant defense responses. NLP-like proteins are considered to be effector proteins, and they have been isolated from bacteria, fungi, and oomycete plant pathogens. There is increasing evidence that NLPs have the ability to induce cell death and ethylene accumulation in plants.ResultsWe evaluated the expression patterns of 11 targeted PcNLP genes by qRT-PCR at different time points after infection by P. capsici. Several PcNLP genes were strongly expressed at the early stages in the infection process, but the expression of other PcNLP genes gradually increased to a maximum at late stages of infection. The genes PcNLP2, PcNLP6 and PcNLP14 showed the highest expression levels during infection by P. capsici. The necrosis-inducing activity of all targeted PcNLP genes was evaluated using heterologous expression by PVX agroinfection of Capsicum annuum and Nicotiana benthamiana and by Western blot analysis. The members of the PcNLP family can induce chlorosis or necrosis during infection of pepper and tobacco leaves, but the chlorotic or necrotic response caused by PcNLP genes was stronger in pepper leaves than in tobacco leaves. Moreover, PcNLP2, PcNLP6, and PcNLP14 caused the largest chlorotic or necrotic areas in both host plants, indicating that these three genes contribute to strong virulence during infection by P. capsici. This was confirmed through functional evaluation of their silenced transformants. In addition, we further verified that four conserved residues are putatively active sites in PcNLP1 by site-directed mutagenesis.ConclusionsEach targeted PcNLP gene affects cells or tissues differently depending upon the stage of infection. Most PcNLP genes could trigger necrotic or chlorotic responses when expressed in the host C. annuum and the non-host N. benthamiana. Individual PcNLP genes have different phytotoxic effects, and PcNLP2, PcNLP6, and PcNLP14 may play important roles in symptom development and may be crucial for virulence, necrosis-inducing activity, or cell death during infection by P. capsici.