Use of a Secretion Trap Screen in Pepper Following Phytophthora capsici Infection Reveals Novel Functions of Secreted Plant Proteins in Modulating Cell Death

In plants, the primary defense against pathogens is mostly inducible and associated with cell wall modification and defense-related gene expression, including many secreted proteins. To study the role of secreted proteins, a yeast-based signal-sequence trap screening was conducted with the RNA from Phytophthora capsici-inoculated root of Capsicum annuum ‘Criollo de Morelos 334’ (CM334). In total, 101 Capsicum annuum secretome (CaS) clones were isolated and identified, of which 92 were predicted to have a secretory signal sequence at their N-terminus. To identify differences in expressed CaS genes between resistant and susceptible cultivars of pepper, reverse Northern blots and real-time reverse-transcription polymerase chain reaction were performed with RNA samples isolated at different time points following P. capsici inoculation. In an attempt to assign biological functions to CaS genes, we performed in planta knock-down assays using the Tobacco rattle virus-based gene-silencing method. Silencing of eight CaS genes in pepper resulted in suppression of the cell death induced by the non-host bacterial pathogen (Pseudomonas syringae pv. tomato T1). Three CaS genes induced phenotypic abnormalities in silenced plants and one, CaS259 (PR4-l), caused both cell death suppression and perturbed phenotypes. These results provide evidence that the CaS genes may play important roles in pathogen defense as well as developmental processes.

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Tipos de compatibilidad A1 y A2 de Phytophthora capsici y P. drechsleri coexisten en plantas ornamentales de vivero

Revista Mexicana de Fitopatología Mexican Journal of Phytopathology ◽

10.18781//r.mex.fit.1712-5 ◽

2018 ◽

Vol 36 (2) ◽

Author(s):

Alejandro Soto-Plancarte ◽

Sylvia Patricia Fernández-Pavía ◽

Gerardo Rodríguez-Alvarado ◽

Luis López-Pérez ◽

Yolanda Leticia Fernández-Pavía ◽

...

Keyword(s):

Capsicum Annuum ◽

Phytophthora Capsici ◽

In Planta ◽

Petunia X Hybrida

No se ha reportado la presencia de ambos tipos de compatibilidad de especies de Phytophthora en plantas hospedantes sembradas en una misma maceta en viveros comerciales de México. En 2015, se colectaron aislados de Phytophthora en viveros comerciales en la Ciudad de México y en el estado de Morelos. Se detectaron los tipos de compatibilidad A1 y A2 de Phytophthora capsici y P. drechsleri en plantas marchitas de Capsicum annuum (tallos) y Petunia x hybrida (rizósfera), respectivamente. Los aislados de Phytophthora coinoculados in planta formaron oosporas en condiciones de invernadero. Phytophthora fue re-aislada de plantas inoculadas e identificada mediante herramientas morfológicas y moleculares. Los tipos de compatibilidad A1 y A2 de Phytophthora capsici y P. drechsleri se presentan simultáneamente en plantas de invernadero, lo cual sugiere que están ocurriendo recombinación sexual y variación genética. Los tipos de compatibilidad A1 y A2 de Phytophthora capsici y Phytophtora dechsleri fueron detectados por primera vez en Capsicum annuum y Petunia x hybrida respectivamente, en viveros mexicanos.

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Characterization of CRN-Like Genes From Plasmopara viticola: Searching for the Most Virulent Ones

Frontiers in Microbiology ◽

10.3389/fmicb.2021.632047 ◽

2021 ◽

Vol 12 ◽

Author(s):

Gaoqing Xiang ◽

Xiao Yin ◽

Weili Niu ◽

Tingting Chen ◽

Ruiqi Liu ◽

...

Keyword(s):

Cell Death ◽

Downy Mildew ◽

Molecular Mechanisms ◽

Phytophthora Capsici ◽

Sequence Divergence ◽

Plasmopara Viticola ◽

Wine Industry ◽

In Planta ◽

Vitis Riparia ◽

Grapevine Downy Mildew

Grapevine downy mildew is an insurmountable disease that endangers grapevine production and the wine industry worldwide. The causal agent of the disease is the obligate biotrophic oomycete Plasmopara viticola, for which the pathogenic mechanism remains largely unknown. Crinkling and necrosis proteins (CRN) are an ancient class of effectors utilized by pathogens, including oomycetes, that interfere with host plant defense reactions. In this study, 27 CRN-like genes were cloned from the P. viticola isolate YL genome, hereafter referred to as PvCRN genes, and characterized in silico and in planta. PvCRN genes in ‘YL’ share high sequence identities with their ortholog genes in the other three previously sequenced P. viticola isolates. Sequence divergence among the genes in the PvCRN family indicates that different PvCRN genes have different roles. Phylogenetic analysis of the PvCRN and the CRN proteins encoded by genes in the P. halstedii genome suggests that various functions might have been acquired by the CRN superfamily through independent evolution of Plasmopara species. When transiently expressed in plant cells, the PvCRN protein family shows multiple subcellular localizations. None of the cloned PvCRN proteins induced hypersensitive response (HR)-like cell death on the downy mildew-resistant grapevine Vitis riparia. This was in accordance with the result that most PvCRN proteins, except PvCRN11, failed to induce necrosis in Nicotiana benthamiana. Pattern-triggered immunity (PTI) induced by INF1 was hampered by several PvCRN proteins. In addition, 15 PvCRN proteins prevented Bax-induced plant programmed cell death. Among the cell death-suppressing members, PvCRN17, PvCRN20, and PvCRN23 were found to promote the susceptibility of N. benthamiana to Phytophthora capsici, which is a semi-biotrophic oomycete. Moreover, the nucleus-targeting member, PvCRN19, promoted the susceptibility of N. benthamiana to P. capsici. Therefore, these PvCRN proteins were estimated to be virulent effectors involved in the pathogenicity of P. viticola YL. Collectively, this study provides comprehensive insight into the CRN effector repertoire of P. viticola YL, which will help further elucidate the molecular mechanisms of the pathogenesis of grapevine downy mildew.

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Variabilidad morfológica y sensibilidad de Phytophthora capsici causando marchitez en chile pimiento morrón en Chihuahua, México

Revista Mexicana de Fitopatología Mexican Journal of Phytopathology ◽

10.18781/r.mex.fit.1904-4 ◽

2019 ◽

Vol 37 (1) ◽

Author(s):

Celina Sánchez-Gurrola ◽

Nuria Gómez-Dorantes ◽

Gerardo Rodríguez-Alvarado ◽

Sylvia Patricia Fernández-Pavía ◽

Graciela Ávila-Quezada

Keyword(s):

Capsicum Annuum ◽

Phytophthora Capsici ◽

In Planta

A nivel mundial, México es el principal exportador del chile pimiento morrón (Capsicum annuum). El cultivo es afectado por diversas enfermedades, siendo la marchitez del chile ocasionada por Phytophthora capsici una de las más importantes. Este fitopatógeno posee alta variabilidad en virulencia y sensibilidad a fungicidas. No existen en México estudios sobre poblaciones de P. capsici para pimiento morrón. El objetivo de este trabajo fue determinar la diversidad de aislados de P. capsici obtenidos de plantas de pimiento morrón con marchitez en Delicias, Chihuahua, México, mediante los marcadores fenotípicos: patrón de crecimiento de la colonia, tipo de compatibilidad, virulencia y sensibilidad a fungicidas. Los tipos de colonia fueron: estrellado (74%), ligeramente petaloide (14%) y radial (12%). Se detectaron aislados sensibles (53%), con sensibilidad intermedia (42%) e insensibles (5%) a mefenoxam. El 86.6% de los aislados inoculados fueron altamente virulentos, produjeron síntomas cuatro días después de la inoculación. Se detectaron los dos tipos de compatibilidad (TC) A1 y A2. Se formaron oosporas in planta al inocular los dos TC detectados en una misma planta. Los resultados sugieren que existe reproducción sexual en campo y como consecuencia variabilidad entre los aislados de P. capsici, responsables de la marchitez en cultivos de chile pimiento morrón en Delicias, Chihuahua, México.

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An Arabidopsis downy mildew non-RxLR effector suppresses induced plant cell death to promote biotroph infection

Journal of Experimental Botany ◽

10.1093/jxb/eraa472 ◽

2020 ◽

Author(s):

Florian Dunker ◽

Lorenz Oberkofler ◽

Bernhard Lederer ◽

Adriana Trutzenberg ◽

Arne Weiberg

Keyword(s):

Cell Death ◽

Pseudomonas Syringae ◽

Plant Cell ◽

Phytophthora Capsici ◽

Ectopic Expression ◽

Molecular Evidence ◽

Disease Symptoms ◽

Rxlr Effectors ◽

Rxlr Effector ◽

Local Plant

Abstract Our understanding of obligate biotrophic pathogens is limited by lack of knowledge concerning the molecular function of virulence factors. We established Arabidopsis host-induced gene silencing (HIGS) to explore gene functions of Hyaloperonospora arabidopsidis, including CYSTEINE-RICH PROTEIN (HaCR)1, a potential secreted effector gene of this obligate biotrophic pathogen. HaCR1 HIGS resulted in H. arabidopsidis-induced local plant cell death and reduced pathogen reproduction. We functionally characterized HaCR1 by ectopic expression in Nicotiana benthamiana. HaCR1 was capable of inhibiting effector-triggered plant cell death. Consistent with this, HaCR1 expression in N. benthamiana led to stronger disease symptoms caused by the hemibiotrophic oomycete pathogen Phytophthora capsici, but reduced disease symptoms caused by the necrotrophic fungal pathogen Botrytis cinerea. Expressing HaCR1 in transgenic Arabidopsis confirmed higher susceptibility to H. arabidopsidis and to the bacterial hemibiotrophic pathogen Pseudomonas syringae. Increased H. arabidopsidis infection was in accordance with reduced PATHOGENESIS RELATED (PR)1 induction. Expression of full-length HaCR1 was required for its function, which was lost if the signal peptide was deleted, suggesting its site of action in the plant apoplast. This study provides phytopathological and molecular evidence for the importance of this widespread, but largely unexplored class of non-RxLR effectors in biotrophic oomycetes.

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Tipos de compatibilidad A1 y A2 de Phytophthora capsici y P. drechsleri coexisten en plantas ornamentales de vivero

Revista Mexicana de Fitopatología Mexican Journal of Phytopathology ◽

10.18781/r.mex.fit.1712-5 ◽

2018 ◽

Vol 36 (2) ◽

Author(s):

Alejandro Soto-Plancarte ◽

Sylvia Patricia Fernández-Pavía ◽

Gerardo Rodríguez-Alvarado ◽

Luis López-Pérez ◽

Yolanda Leticia Fernández-Pavía ◽

...

Keyword(s):

Capsicum Annuum ◽

Phytophthora Capsici ◽

In Planta ◽

Petunia X Hybrida

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Nuclear Localization of HopA1Pss61 Is Required for Effector-Triggered Immunity

Plants ◽

10.3390/plants10050888 ◽

2021 ◽

Vol 10 (5) ◽

pp. 888

Author(s):

Hobin Kang ◽

Quang-Minh Nguyen ◽

Arya Bagus Boedi Iswanto ◽

Jong Chan Hong ◽

Saikat Bhattacharjee ◽

...

Keyword(s):

Cell Death ◽

Pseudomonas Syringae ◽

Nuclear Localization ◽

Functional Characterization ◽

Defense Responses ◽

Disease Resistance Gene ◽

In Planta ◽

Resistance Proteins ◽

Cell Death Response ◽

Effector Triggered Immunity

Plant resistance proteins recognize cognate pathogen avirulence proteins (also named effectors) to implement the innate immune responses called effector-triggered immunity. Previously, we reported that hopA1 from Pseudomonas syringae pv. syringae strain 61 was identified as an avr gene for Arabidopsis thaliana. Using a forward genetic screen approach, we cloned a hopA1-specific TIR-NBS-LRR class disease resistance gene, RESISTANCE TO PSEUDOMONAS SYRINGAE6 (RPS6). Many resistance proteins indirectly recognize effectors, and RPS6 is thought to interact with HopA1Pss61 indirectly by surveillance of an effector target. However, the involved target protein is currently unknown. Here, we show RPS6 is the only R protein that recognizes HopA1Pss61 in Arabidopsis wild-type Col-0 accession. Both RPS6 and HopA1Pss61 are co-localized to the nucleus and cytoplasm. HopA1Pss61 is also distributed in plasma membrane and plasmodesmata. Interestingly, nuclear localization of HopA1Pss61 is required to induce cell death as NES-HopA1Pss61 suppresses the level of cell death in Nicotiana benthamiana. In addition, in planta expression of hopA1Pss61 led to defense responses, such as a dwarf morphology, a cell death response, inhibition of bacterial growth, and increased accumulation of defense marker proteins in transgenic Arabidopsis. Functional characterization of HopA1Pss61 and RPS6 will provide an important piece of the ETI puzzle.

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VipariNama: RNA viral vectors to rapidly elucidate the relationship between gene expression and phenotype

Plant Physiology ◽

10.1093/plphys/kiab197 ◽

2021 ◽

Author(s):

Arjun Khakhar ◽

Cecily Wang ◽

Ryan Swanson ◽

Sydney Stokke ◽

Furva Rizvi ◽

...

Keyword(s):

Gene Expression ◽

Transcription Factors ◽

Viral Vectors ◽

Viral Vector ◽

Tobacco Rattle Virus ◽

Plant Size ◽

Great Promise ◽

Attractive Alternative ◽

Gibberellin Biosynthesis ◽

In Planta

Abstract Synthetic transcription factors have great promise as tools to help elucidate relationships between gene expression and phenotype by allowing tunable alterations of gene expression without genomic alterations of the loci being studied. However, the years-long timescales, high cost, and technical skill associated with plant transformation have limited their use. In this work we developed a technology called VipariNama (ViN) in which vectors based on the Tobacco Rattle Virus (TRV) are used to rapidly deploy Cas9-based synthetic transcription factors and reprogram gene expression in planta. We demonstrate that ViN vectors can implement activation or repression of multiple genes systemically and persistently over several weeks in Nicotiana benthamiana, Arabidopsis (Arabidopsis thaliana), and tomato (Solanum lycopersicum). By exploring strategies including RNA scaffolding, viral vector ensembles, and viral engineering, we describe how the flexibility and efficacy of regulation can be improved. We also show how this transcriptional reprogramming can create predictable changes to metabolic phenotypes, such as gibberellin biosynthesis in N. benthamiana and anthocyanin accumulation in Arabidopsis, as well as developmental phenotypes, such as plant size in N. benthamiana, Arabidopsis, and tomato. These results demonstrate how ViN vector-based reprogramming of different aspects of gibberellin signaling can be used to engineer plant size in a range of plant species in a matter of weeks. In summary, VipariNama accelerates the timeline for generating phenotypes from over a year to just a few weeks, providing an attractive alternative to transgenesis for synthetic transcription factor-enabled hypothesis testing and crop engineering.

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Uncoupling Salicylic Acid-Dependent Cell Death and Defense-Related Responses From Disease Resistance in the Arabidopsis Mutant acd5

Genetics ◽

10.1093/genetics/156.1.341 ◽

2000 ◽

Vol 156 (1) ◽

pp. 341-350

Author(s):

Jean T Greenberg ◽

F Paul Silverman ◽

Hua Liang

Keyword(s):

Cell Death ◽

Salicylic Acid ◽

Disease Resistance ◽

Pseudomonas Syringae ◽

Higher Plants ◽

Ethylene Signaling ◽

Symptom Development ◽

Wild Type ◽

Dependent Cell ◽

Arabidopsis Mutant

Abstract Salicylic acid (SA) is required for resistance to many diseases in higher plants. SA-dependent cell death and defense-related responses have been correlated with disease resistance. The accelerated cell death 5 mutant of Arabidopsis provides additional genetic evidence that SA regulates cell death and defense-related responses. However, in acd5, these events are uncoupled from disease resistance. acd5 plants are more susceptible to Pseudomonas syringae early in development and show spontaneous SA accumulation, cell death, and defense-related markers later in development. In acd5 plants, cell death and defense-related responses are SA dependent but they do not confer disease resistance. Double mutants with acd5 and nonexpressor of PR1, in which SA signaling is partially blocked, show greatly attenuated cell death, indicating a role for NPR1 in controlling cell death. The hormone ethylene potentiates the effects of SA and is important for disease symptom development in Arabidopsis. Double mutants of acd5 and ethylene insensitive 2, in which ethylene signaling is blocked, show decreased cell death, supporting a role for ethylene in cell death control. We propose that acd5 plants mimic P. syringae-infected wild-type plants and that both SA and ethylene are normally involved in regulating cell death during some susceptible pathogen infections.

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Zinc phosphate protects tomato plants against Pseudomonas syringae pv. tomato

Journal of Plant Diseases and Protection ◽

10.1007/s41348-021-00444-z ◽

2021 ◽

Author(s):

Mara Quaglia ◽

Marika Bocchini ◽

Benedetta Orfei ◽

Roberto D’Amato ◽

Franco Famiani ◽

...

Keyword(s):

Disease Severity ◽

Pseudomonas Syringae ◽

Zinc Phosphate ◽

Plant Defence ◽

In Planta ◽

Tomato Plants ◽

Pathogenesis Related Protein ◽

Defence Responses ◽

Plant Defence Responses

AbstractThe purpose of this study was to determine whether zinc phosphate treatments of tomato plants (Solanum lycopersicum L.) can attenuate bacterial speck disease severity through reduction of Pseudomonas syringae pv. tomato (Pst) growth in planta and induce morphological and biochemical plant defence responses. Tomato plants were treated with 10 ppm (25.90 µM) zinc phosphate and then spray inoculated with strain DAPP-PG 215, race 0 of Pst. Disease symptoms were recorded as chlorosis and/or necrosis per leaf (%) and as numbers of necrotic spots. Soil treatments with zinc phosphate protected susceptible tomato plants against Pst, with reductions in both disease severity and pathogen growth in planta. The reduction of Pst growth in planta combined with significantly higher zinc levels in zinc-phosphate-treated plants indicated direct antimicrobial toxicity of this microelement, as also confirmed by in vitro assays. Morphological (i.e. callose apposition) and biochemical (i.e., expression of salicylic-acid-dependent pathogenesis-related protein PR1b1 gene) defence responses were induced by the zinc phosphate treatment, as demonstrated by histochemical and qPCR analyses, respectively. In conclusion, soil treatments with zinc phosphate can protect tomato plants against Pst attacks through direct antimicrobial activity and induction of morphological and biochemical plant defence responses.

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Prediction and Activity of a Cationic α-Helix Antimicrobial Peptide ZM-804 from Maize

International Journal of Molecular Sciences ◽

10.3390/ijms22052643 ◽

2021 ◽

Vol 22 (5) ◽

pp. 2643

Author(s):

Mohamed F. Hassan ◽

Abdelrahman M. Qutb ◽

Wubei Dong

Keyword(s):

Cdna Library ◽

Pseudomonas Syringae ◽

Inbred Line ◽

Mammalian Cells ◽

Laser Scanning ◽

Pathogenic Bacteria ◽

Antimicrobial Activities ◽

Confocal Laser ◽

In Planta ◽

Minimal Inhibitory Concentrations

Antimicrobial peptides (AMPs) are small molecules consisting of less than fifty residues of amino acids. Plant AMPs establish the first barrier of defense in the innate immune system in response to invading pathogens. The purpose of this study was to isolate new AMPs from the Zea mays L. inbred line B73 and investigate their antimicrobial activities and mechanisms against certain essential plant pathogenic bacteria. In silico, the Collection of Anti-Microbial Peptides (CAMPR3), a computational AMP prediction server, was used to screen a cDNA library for AMPs. A ZM-804 peptide, isolated from the Z. mays L. inbred line B73 cDNA library, was predicted as a new cationic AMP with high prediction values. ZM-804 was tested against eleven pathogens of Gram-negative and Gram-positive bacteria and exhibited high antimicrobial activities as determined by the minimal inhibitory concentrations (MICs) and the minimum bactericidal concentrations (MBCs). A confocal laser scanning microscope observation showed that the ZM-804 AMP targets bacterial cell membranes. SEM and TEM images revealed the disruption and damage of the cell membrane morphology of Clavibacter michiganensis subsp. michiganensis and Pseudomonas syringae pv. tomato (Pst) DC3000 caused by ZM-804. In planta, ZM-804 demonstrated antimicrobial activity and prevented the infection of tomato plants by Pst DC3000. Moreover, four virulent phytopathogenic bacteria were prevented from inducing hypersensitive response (HR) in tobacco leaves in response to low ZM-804 concentrations. ZM-804 exhibits low hemolytic activity against mouse red blood cells (RBCs) and is relatively safe for mammalian cells. In conclusion, the ZM-804 peptide has a strong antibacterial activity and provides an alternative tool for plant disease control. Additionally, the ZM-804 peptide is considered a promising candidate for human and animal drug development.

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