Plant Resistance to Root-Knot Nematodes through RNA Interference Targeting a Parasitism Gene

Schweri, Kathryn Keller North Carolina State University 2014 해외박사(DDOD)

소속기관이 구독 중이 아닌 경우 오후 4시부터 익일 오전 9시까지 원문보기가 가능합니다.

Root-knot nematodes (RKN), members of the genus Meloidogyne, are sedentary plant-parasitic nematodes that infect multiple crop species and cause staggering economic losses worldwide. Proteins produced in specialized esophageal gland cells are secreted from the stylet of RKN to transform recipient host plant root cells into multinucleate giant-cells that are essential for nematode feeding. The peptide encoded by the Meloidogyne incognita 16D10 parasitism gene has previously been shown to be involved in giant-cell formation, and host-derived RNA interference (RNAi) against the 16D10 transcript in Arabidopsis thaliana resulted in resistance to the four major species of RKN (Huang et al., 2006b). The first objective of this dissertation was to transform two host crop species, tobacco and strawberry, with 16D10RNAi constructs and to test for resistance of the resulting transgenic plants to RKN species. Haploid plants of the Nicotiana tabacum cultivars TN90 (burley) and Hicks (flue-cured) were transformed with the 16D10RNAi constructs used by Huang et al. (2006b), and doubled haploid plants were produced. Expression of siRNA by the transgenic tobacco was confirmed by RT-PCR and high-throughput siRNA Ion Proton sequencing. Several lines of the 16D10RNAi transgenic tobacco were found to be significantly more resistant to root-knot nematodes than wild-type untransformed controls. A maximum of 62% reduction in Meloidogyne arenaria egg production and a maximum of 52% reduction in M. incognita egg production were observed in TN90 16D10RNAi tobacco roots, and a maximum of 73% reduction in egg production of M. arenaria was observed in roots of Hicks 16D10RNAi tobacco plants. Transformation of strawberry with the 16D10-RNAi constructs was also attempted, however explant browning in tissue culture prevented the production of regenerated transgenic strawberry plants. The research described here addresses some of the challenges in strawberry plant tissue culture, and suggests methods for overcoming these issues. The second objective of this research was to design and create new 16D10 siRNA expression vectors in an attempt to improve siRNA expression. Currently, all work with 16D10RNAi has been performed using the pHANNIBAL vector (Wesley et al., 2001), which utilizes the CaMV 35S promoter and the PDK intron as a spacer. The 16D10RNAi-expressing tobacco using the pHANNIBAL vector was found to be less resistant to root-knot nematodes than the 16D10RNAi Arabidopsis created by Huang et al. (2006b). The purpose of this research was to determine if altering the 16D10RNAi expression constructs could produce greater siRNA expression and potentially greater resistance to root-knot nematodes. The 35S promoter was exchanged for two new promoters, Gmubi and Ntcel7, and the GUS spacer was also substituted for the PDK intron to improve hairpin double-stranded RNA intron splicing. Gmubi is a constitutive promoter like 35S, but has been shown to result in greater expression than 35S in soybean (Chiera et al. 2007). The Ntcel7 promoter is a tissue-specific promoter that is only expressed in shoot and root meristematic tissue and weakly expressed in the vasculature, but has been shown to be up-regulated in giant-cells (Wang et al., 2007). All vectors were successfully assembled and electroporated into Agrobacterium tumefaciens for plant transformations. To test the efficacy of the new constructs, they were all transformed into Arabidopsis thaliana using the floral dip method. Self-pollination and selection of Arabidopsis to obtain homozygous lines is currently underway and the resulting lines will be tested for siRNA expression and root-knot nematode resistance.

Physiological and molecular analysis of dnd "defense, no death" mutants of Arabidopsis

Smith, Roger Kenneth, Jr University of Illinois at Urbana-Champaign 2002 해외박사(DDOD)

소속기관이 구독 중이 아닌 경우 오후 4시부터 익일 오전 9시까지 원문보기가 가능합니다.

Plant diseases cause billions of dollars in damage to crops annually, making dissection of the molecular mechanisms of plant disease resistance an economically important task. Gene-for-gene resistance mediated by plant <italic> R</italic> genes and pathogen <italic>avr</italic> genes has long been associated with the hypersensitive response (HR). Previously, <italic>Arabidopsis thaliana </italic> “defense, no death” (<italic>dnd</italic>) mutants were identified based on their failure to produce the HR when inoculated with <italic> P. syringae</italic> carrying the avirulence gene <italic>avrRpt2</italic>. The first objective of this study was to characterize the <italic>dnd2</italic> mutants in relation to defense. Like the previously characterized <italic> dnd1</italic> mutant plants, <italic>dnd2</italic> mutants responded to avirulent pathogens by activating <italic>avr</italic>-dependent defenses despite the absence of the HR. In addition, <italic>dnd2</italic> plants exhibited a constitutive systemic resistance associated with elevated levels of salicylic acid (SA). SA was found to be required for the systemic resistance of <italic>dnd2</italic> plants, but was not necessary for the HR<super>−</super> phenotype displayed by the mutant plants. SA-independent pathways not normally activated by <italic> P. syringae</italic> infection were activated in <italic>dnd</italic> plants, as indicated by the marker for SA-independent signaling, PDF 1.2. A map-based cloning project was initiated which identified a precise genetic interval spanning <italic>DND2</italic>. Focused shotgun complementation experiments were initiated using subclones from within this region. The roles of reactive oxygen species (ROS) and the enzymes that scavenge these signaling molecules were examined in relation to the HR<super>−</super> phenotype of <italic> dnd</italic> plants. Northern analysis of ROS scavenger mRNA revealed no significant differences in gene expression between wild type and <italic>dnd</italic> plants, suggesting that ROS scavengers are not blocking ROS signaling for the HR. Finally, the dwarfed stature of <italic>dnd</italic> plants was could be partially corrected by altered growth conditions, specifically the soil mix. Chemical analysis of soil mixes after 6 weeks of use suggested that cations and pH might be involved in the growth phenotype. The effect of K<super>+ </super>, Ca<super>2+</super>, Na<super>+</super>, and pH on <italic>dnd</italic> plant growth was then tested in agar-based media. The <italic>dnd</italic> plants did not respond to increasing concentrations of Ca<super>2+</super> in parallel with wild-type plants. K<super>+</super>, Na<super>+</super> and pH impacted <italic>dnd1, dnd2</italic>, and wild-type plants similarly.

Improving Abiotic and Biotic Stress Tolerance in Floriculture Crops

South, Kaylee Anne The Ohio State University ProQuest Dissertations & 2020 해외박사(DDOD)

소속기관이 구독 중이 아닌 경우 오후 4시부터 익일 오전 9시까지 원문보기가 가능합니다.

An intensive production system is used to produce greenhouse floriculture crops, marketed for their flowers and attractive foliage. Chemical, environmental, and cultural methods are used to manage biotic and abiotic stresses during production. Additional tools are needed by growers because of growing concerns around the negative impact of plant production on humans and the environment. The objective of this research was to evaluate potential tools to improve floriculture crop resilience under stress during production and post-production.Botrytis cinerea causes disease in most major greenhouse crops and is resistant to several fungicides. Additional control methods, like plant growth promoting bacteria (PGPB) that can improve plant performance by increasing plant resilience to stress are needed. A collection of 60 bacterial strains was evaluated in a dual culture assay and an initial greenhouse trial with Petunia x hybrida Carpet Red Bright’ to identify strains for the biocontrol of B. cinerea. Daily flower disease severity ratings were used to select seven strains that were evaluated in the validation greenhouse trial. Three Pseudomonas strains were selected for the greatest reduction in B. cinerea infection.The efficacy of PGPB and the plant’s susceptibility to B. cinerea were affected by fertilization. Petunia x hybrida `Carpet Red Bright’ was treated with bacteria or a commercial biocontrol product and fertilized with synthetic chemical or organic fertilizer at a low or high rate. Measured plant growth and flower disease severity revealed that plants with the high rate synthetic fertilizer were the largest and had the lowest disease severity. Reduction of disease severity varied between bacterial and fertilizer treatment combinations. Plants treated with one bacterium had reduced disease severity at the high rate synthetic chemical fertilizer but not at the low rate organic fertilizer.Specific fertility programs provide crops with needed macro and micronutrients, but overuse can lead to negative environmental impacts, plant disorders, and higher susceptibility to other stresses. Application of PGPB can improve floriculture plant performance grown under low fertility. Ninety-four bacterial isolates identified from the rhizosphere of ornamental plants were evaluated in P. hybrida `Picobella Blue’ grown under low fertility, and 15 isolates were selected for increasing plant performance. Whole-genome sequencing was used to determine their identity and bacteria were evaluated again under low fertility along with untreated plants receiving higher fertilizer rates. Three bacteria were selected as top performers for increases in flowering, vegetative health, and vegetative quality.Over- or under-watering is a common stress for Phalaenopsis orchids once the orchid reaches the consumer, but to avoid water stress, ice cube irrigation is recommended. Orchid health was evaluated after irrigation with either ice cubes or room temperature water. Orchids grown in bark media did not have a reduction in display life or health when irrigated with ice cubes.Utilizing novel tools, like PGPB or ice cube irrigation, during production or post-production is an important move toward improving floriculture crop performance. These tools can be used to improve floriculture crop resilience to biotic or abiotic stresses and establish additional sustainable practices for the greenhouse industry.

The Role of Terrestrial Mollusks in Phoresis and Vectoring of Plant-Parasites

Sanchez, Kristi Rosanne University of California, Davis 2015 해외박사(DDOD)

소속기관이 구독 중이 아닌 경우 오후 4시부터 익일 오전 9시까지 원문보기가 가능합니다.

The Brown Garden Snail, Helix aspersa, is a terrestrial mollusk that was introduced to California in the 1850s, and many other parts of the world as food (escargot), through the movement of plants, and by hobbyists who collect snails. Today, H. aspersa is a pest that can be found almost anywhere within California. This research determined the nematodes, fungi, and bacteria associated with H. aspersa collected in three locations in San Diego, California, and assessed where on or in the snail body the microbes were carried. All the microbes recovered from individual snails were identified as operational taxonomic units (OTUs) through comparison of their DNA sequence homology with available data. Twenty-three nematode operational taxonomic units (OTUs) were recovered from the snails. Bacterivorous nematodes recovered included Caenorhabitis elegans, Rhabditis sp., and Panagrolaimus species. Plant parasitic nematode OTUs included Diytlenchus dipsaci and Pratylenchus species. The bacterial and fungal OTUs isolated included Pseudomonas putida, and Stentrophomonas maltophilia, and fungal plant pathogens Fusarium solani, F. oxysporum f. sp. chrysanthemi, Rhizoctonia solani. Laboratory and growth chamber experiments were conducted to determine whether plant-pathogens inoculum is potentially disseminated and transmitted by H. aspersa. In laboratory experiments, individual snails were fed inoculum of Fusarium circinatum and Rhizoctonia solani as preserved mycelia on filter paper, with control snails being fed sterile filter paper. After H. aspersa consumed the filter paper with pathogen inoculum, viable fungus was recovered from 90% of fecal pellets for up to 9 days post feeding, while no pathogens were detected control snail fecal pellets. Vermiform, active plant-parasitic nematodes of Meloidogyne hapla, Diytlenchus dipsaci, and Aphelenchoides ritzemabosi were fed to snails as an aqueous nematode suspension placed on the surface of a single carrot disc (1.5 g). Viable vermiform nematodes of all the tested species were recovered from the fecal pellets of the snails post feeding. The nematodes passed through the snail digestive tract and recovered from fecal pellets over a time period of 2-8 days post feeding. In growth chamber experiments, active second-stage juveniles (J2) of root-knot nematodes M. hapla and M. incognita were fed to snails as an aqueous nematode suspension (a total of 1000 J2s) placed on the surface of a single carrot disc (1.5 g). Control snails received carrots without nematode inoculum. Subsequent to feeding H. aspersa the J2s, fecal pellets were recovered and placed at the base of individual tomato seedlings growing in a growth chamber. After two months, the tomato plants were destructively sampled and assessed for root-knot nematode infection. Control plants were not infected, but the plants exposed to fecal pellets from snails fed the root-knot nematode J2s were infected, showing symptoms of galled roots and erioglaucine-stained egg masses visible on the roots. A diverse range of nematode, fungal, and bacterial OTUs were recovered from field-collected H. aspersa in California. Many of the recovered OTUs were plant pathogens, and inoculum propagules of fungal and nematode plant pathogens can transit the H. aspersa digestive system in viable condition. Second-stage juveniles of the root-knot nematodes M. hapla and M. incognita survive transit of the H. aspersa digestive system, and are infective; being able to emerge from fecal pellets, move into soil, find host roots, and establish successful infections and reproduce. Plant pathogens are not infrequent associates of H. aspersa, and can potentially aid in pathogen dispersal and transmission. This new information should prompt further consideration of management and monitoring of mollusk pests in California agriculture.

Studies of a TMV-based expression vector in plant cell cultures

Shiratori, Masaru Ken University of California, Davis 2004 해외박사(DDOD)

소속기관이 구독 중이 아닌 경우 오후 4시부터 익일 오전 9시까지 원문보기가 가능합니다.

Plant virus expression vectors for transient heterologous protein production have been developed and successfully used in whole plant host expression systems. However, there is currently no published work concerning the development of plant virus expression vectors in plant cell culture. We established and studied cell cultures infected with a TMV-based expression vector, 30Bcycle3GFP, containing the gene for Cycle3GFP under the control of a strong promoter. The infected cultures were derived from a variety of methods. First, we initiated infected cell cultures from plants infected with 30Bcycle3GFP. Second, 30Bcycle3GFP infected cell cultures were obtained using a direct inoculation technique. Third, infected cell cultures showing antibiotic resistance were developed by co-culturing infected cell cultures with transgenic cell cultures constitutively expressing a gene product for antibiotic resistance. Additionally, we examined the possibility of developing infected cell cultures from high-efficiency protoplast transfections with 30Bcycle3GFP. The production of Cycle3GFP, levels of viral vector RNA, and the insertional stability of the Cycle3GFP gene were characterized in the infected cultures. We obtained fluorescing Cycle3GFP levels as high as 2.4% of the total soluble proteins (%TSP) extracted from infected cultures initiated from infected plants. In directly inoculated cell cultures, we achieved Cycle3GFP levels as high as 0.7% TSP extracted although infection efficiencies were low. Maximum Cycle3GFP expression levels of 4.9% TSP extracted were obtained in infected cultures exhibiting antibiotic resistance. Derivatives of the 30Bcycle3GFP vector that had lost the Cycle3GFP gene were found in several infected cultures and we provide evidence that some vector derivatives were replicating. There were no detectable levels of 30Bcycle3GFP vectors showing loss of insert in directly inoculated cultures examined one month post-inoculation. In addition, some infected cultures maintained Cycle3GFP fluorescence for generations with manual selection of fluorescing tissues during subculturings. Our data suggest that a selection process for viral vector infected cells would increase the stability of heterologous protein expression and reduce the heterogeneity of viral vector infection in our system. Furthermore, the ability to directly inoculate plant cell cultures with a viral vector shows the potential for a rapid, high-level heterologous protein expression system in plant cell culture.

Molecular, Proteomics and Cytochemical Analyses of the PR 10-LRR Complex, Abscisic Acid Responsive Protein 1, Osmotin-like Protein, Formate Dehydrogenase and Phosphoenolpyruvate Carboxykinase Genes for Cell Death and Defense Responses to Microbial Pathogens in Pepper and Arabidopsis

최두석 고려대학교 대학원 2012 국내박사

Plants have evolved a multitude of defense strategies to combat an abundance of microbial pathogens. Recently, molecular research of plant innate immunity has advanced toward the plant molecular breeding to generate the genetically modified, disease resistant plants. In this study, molecular and biochemical mechanisms underlying plant defense immune responses were analyzed using the pepper (Capsicum annuuum)-Xanthomonas campestris pv. vesicatoria (Xcv) and Arabidopsis (Arabidopsis thaliana)-Pseudomonas syringae pv. tomato (Pst) systems. The pepper defense response genes including Pathogenesis-related protein 10 (CaPR10), Abscisic acid-responsive protein 1 (CaABR1), Osmotin-like protein 1 (CaOSM1), Formate dehydrogenase 1 (CaFDH1), and Phosphoenolpyruvate carboxykinase 1 (CaPEPCK1) have been isolated and identified from the pepper leaves infected with both virulent (Ds1) and avirulent (Bv5-4a) strains of Xcv using the differential hybridization screening, yeast-two-hybrid assay as well as proteomics approach. In addition, Arabidopsis Formate dehydrogenase 1 (AtFDH1) has been isolated from the transgenic Arabidopsis plants overexpressing pepper Pathogen-induced membrane protein 1 (CaPIMP1). Virus-induced gene silencing (VIGS), ectopic gene overexpression, transient in planta expression, and T-DNA insertional mutation were used to investigate the gain-of- and loss-of-functions of the defense response genes in pepper, Arabidopsis and Nicotiana benthamiana plants. CaPR10 triggered hypersensitive cell death response (HR), which was promoted by the formation of the protein complex with a leucine-rich repeat protein (CaLRR1) as a positive regulator. CaABR1, a GRAM (for Glucosyltransferases, Rab-like GTPase activators, and Myotubularins) domain-containing protein, functioned in cell death regulation and abscisic acid (ABA)-salicylic acid (SA) antagonism. Notably, the specific subcellular localization of the CaABR1 protein and the CaPR10-CaLRR1 complex to the nucleus and the cytoplasm, respectively, was essential for their function to induce HR. CaOSM1 was required for the induction of HR and reactive oxygen species (ROS) burst in plant cells. CaFDH1, which catalyzes the oxidation of formate into carbon dioxide in the mitochondria in a NAD+-dependent manner, acted as a positive regulator of cell death response. AtFDH1 also played a distinct role for defense and cell death responses to microbial pathogens. CaPEPCK1 positively regulated plant innate immunity against the hemibiotrophic bacterial Pst and obligate biotrophic oomycete Hyaloperonospora arabidopsidis pathogens. Taken together, these results presented in this study suggest that these defense-related genes in pepper and Arabidopsis are responsible for plant cell death and immunity against microbial pathogens. 식물은 다수의 방어 전략을 진화시켜 수많은 병원미생물들에 대항해오고 있다. 최근에 식물의 선천적 면역성에 대한 분자적 연구가 식물 분자 육종으로 발전하여 유전적으로 변형된 병저항성 식물을 제조하기에 이르렀다. 본 연구에서는 고추식물 (Capsicum annuuum)과 Xanthomonas campestris pv. vesicatoria (Xcv), 애기장대 (Arabidopsis thaliana)와 Pseudomonas syringae pv. tomato (Pst) 시스템을 이용하여 식물 방어 면역 반응에 기초하는 분자적&#8226;생화학적 기작을 분석하였다. Proteomics 접근방법뿐만 아니라 differential hybridization screening, yeast-two-hybrid assay를 이용하여 Xcv 의 병원성 (Ds1) 균주와 비병원성 (Bv5-4a)균주에 감염된 고추 잎에서 Pathogenesis-related protein 10 (CaPR10), Osmotin-like protein 1 (CaOSM1), Abscisic acid-responsive protein 1 (CaABR1), Formate dehydrogenase 1 (CaFDH1), Phosphoenolpyruvate carboxykinase 1 (CaPEPCK1) 유전자를 분리&#8226;동정하였다. 또한 고추의 Pathogen-induced membrane protein 1 (CaPIMP1) 유전자를 과발현하는 형질전환 애기장대식물에서 Arabidopsis Formate dehydrogenase 1 (AtFDH1)를 분리하였다. Virus-induced gene silencing (VIGS), 외부 유전자 과발현, 일시적 in planta 유전자 발현, 그리고 T-DNA 삽입 돌연변이를 이용하여 고추, 애기장대, Nicotiana benthamiana 식물에서 이들 방어반응 유전자의 기능 획득(gain-of-function)과 기능 손실(loss-of-function)을 연구하였다. CaPR10은 과민성 세포사멸을 일으키며 CaLRR1과 단백질 복합체를 형성하여 긍정적 조정자로서 세포사멸을 촉진시켰다. CaABR1은 GRAM (for Glucosyltransferases, Rab-like GTPase activators, and Myotubularins) 도메인을 함유하는 단백질이며 세포사멸조절과 앱시스산-살리실산의 길항작용의 기능을 하였다. 특히, CaABR1와 CaPR10-CaLRR1 복합체가 각각 세포내의 핵(nucleus)과 세포질(cytoplasm)로 특이적으로 위치하는 것이 과민성 세포사멸을 유도하기 위해 필수적이었다. CaOSM1은 식물세포에서 과민성 세포사멸의 유도와 활성산소(ROS) 생성에 필요하였다. 미토콘드리아에서 NAD+ 의존적인 방법으로 formate를 산화시켜 이산화탄소를 만드는 반응을 촉매하는 CaFDH1은 세포 사멸 반응의 긍정적 조절자로서 기능을 하였다. 애기장대의 AtFDH1 역시 병원미생물에 대한 방어와 세포 사멸 반응을 위해 상이한 역할을 하였다. CaPEPCK1은 반활물 세균성 병원균 Pst와 절대기생성 난균류 Hyaloperonospora arabidopsidis 에 대한 식물의 선천적 면역성을 긍정적으로 조절하였다. 종합하면, 본 연구에서 제시된 실험결과들은 고추와 애기장대의 이들 방어관련 유전자들이 병원미생물에 대한 식물의 세포 사멸과 면역성에 관여한다는 것을 시사하고 있다.

Polygalacturonase-inhibiting protein sequence, structural, and functional analyses with implications in plant disease resistance

Chestnut, Zachary Andrew University of California, Davis 2015 해외박사(DDOD)

소속기관이 구독 중이 아닌 경우 오후 4시부터 익일 오전 9시까지 원문보기가 가능합니다.

Plant-pathogen interactions result in degrees of plant resistance or susceptibility depending on, in part, numerous biotic factors from both players. Microbe and/or pest challenges to plants often involve the plant cell wall, an outer compartment with roles in cell support and signaling. This dissertation examines the specific interplay between a class of pathogen virulence factors that can degrade the pectin component of cell walls, polygalacturonases (PGs), and plant proteins that limit PG activity and serve as defense factors, polygalacturonase-inhibiting proteins (PGIPs). PGIPs' role in plant defense response pathways is detailed in Chapter 1. PGIPs are common to all angiosperms examined and have diversified into small gene families in many lineages. The sequence variation of 237 PGIPs from 114 angiosperm species was examined here to identify regions of diversity and to attempt to predict the consequent implications on PGIP structure. The majority of divergent residues lie within the beta-sheet shown to interact with PGs. This variation indicates that structural consideration must be taken when selecting PGIPs for inhibition of specific PGs. Pierce's disease (PD) of grapevines is caused by the bacterium Xylella fastidiosa (Xf), which resides in the xylem vessels, systemically infecting vines and eventually causing plant death. The PG of Xf is necessary for systemic vine colonization. Simulated interaction models of XfPG and various PGIPs highlight residues that help stabilize the complex between the pear fruit PGIP (pPGIP) and XfPG; these same complexes are less electrostatically feasible in reactions modeled with a grape PGIP. Transgenic grapevines expressing pPGIP were previously shown to provide increased PD protection, presumably through XfPG inhibition. We developed recombinant expression systems to compare angiosperm PGIPs' inhibition of XfPG through in vitro and in vivo assays to find an optimal inhibitor to exploit for PD protection. Given that pPGIP expression imparts increased PD resistance in greenhouse trials, we sought to evaluate the pPGIP-expressing grapevines in a commercially relevant setting. Field examinations in two agricultural areas -- Solano and Riverside Counties, CA -- were established to test pPGIP efficacy against mechanical inoculations or natural infections of Xf, respectively. PGIPs, as cell wall proteins, are found in the xylem sap and are, therefore, able to cross graft junctions from PGIP-expressing rootstocks into target scions. We confirmed that pPGIP from transgenic rootstocks can be recovered in scion leaves that do not express pPGIP. Grafted grapevines were tested in the field alongside the own-rooted transgenic and control vines for PD resistance. PD Symptoms at the Solano site appeared after two years of annual mechanical inoculations and were reduced in 'Thompson Seedless' vines expressing pPGIP. Symptoms at the Riverside site show similar trends with pPGIP affording some protection, though unintended diseases have confounded results from that site. The transgrafting strategy used here, delivering defense factors via transgenic rootstocks, has promising commercial applications within the evolving regulatory climate for genetically modified plant products. The phylogenetic and structural analyses of PGIPs detailed in Chapter 2 provide a framework to study the predicted efficacy of a PGIP to inhibit numerous PGs from important pathogens. Such candidate PGIPs can then be examined using the techniques refined in Chapter 3, with promising PGIPs tested in expanded trials, as was started with grapevines and pPGIP in Chapter 4.

Uncovering the mechanisms controlling metal micronutrient homeostasis in plants

Socha, Amanda L Dartmouth College 2016 해외박사(DDOD)

소속기관이 구독 중이 아닌 경우 오후 4시부터 익일 오전 9시까지 원문보기가 가능합니다.

As the human population increases, it is imperative that we develop strategies to improve agricultural productivity to meet the growing demand for food, fuel and fiber. More than 2 billion people suffer from "hidden hunger" (a lack of essential nutrients). Such micronutrient deficiencies are most common in developing countries where diets are primarily plant-based. Iron (Fe), one of the essential micronutrients, plays vital roles in both human and plant health. In plants, it is required for essential cellular processes such as photosynthesis and respiration. However, in excess, Fe can spontaneously produce reactive oxygen species (ROS) via the Fenton reaction. Fe-mediated production of reactive oxygen species (ROS) can be harmful to plant tissues, but is part of the defensive strategy against microbes. This thesis focuses on understanding 1) the role of Fe in the formation of ROS for defense against abiotic stress and 2) how Fe, Mn and Zn are loaded into the developing seed and mobilized following germination. Pretreatment of the model plant Arabidopsis thaliana with Fe significantly enhances plants defense to the bacterial pathogen Pseudomonas syringae PtoDC3000. Conversely, chelating soil iron is detrimental to plant defense. A previously uncharacterized Fe-regulated gene, Induced Systemic Resistance 2 (ISR2), negatively regulates the Fe-mediated ROS formation for defense against Pto DC3000 resulting in enhanced resistance in the loss of function mutants and increased susceptibility in overexpression lines. Furthermore, to better understand how essential nutrients are loaded into seeds, Synchrotron X-Ray Fluorescence was employed to image Fe, Zn and Mn in developing and germinating seeds. Zn and Mn are broadly distributed throughout the outer integument cells of the developing seed coat. In contrast, the majority of Fe is localized to the endosperm and to the vascular bundle cells in the embryo, indicating that the Fe pattern is set up early during development. The Zn and Mn gradients in the seed proper occurs much later in seed development, during the late bent cotyledon stage of development when Mn can now be seen to localize to a single layer of spongy mesophyll cells. After germination, Fe and Mn are found in the palisade mesophyll cells between 32-56 hours after imbibition. The mobilization of Fe and Mn is dependent on the Fe and Mn vacuolar exporters NRAMP3 and NRAMP4. Overall, this work has contributed to our knowledge of the role of Fe in plant defense and identified a key player in the basal plant defense response. In addition, our work characterizing metal phenotypes of developing and germinating seeds has improved our understanding of the path that metals take so that they are properly stored and accessed in the seed.

Factors Contributing to Abscisic Acid-Mediated Predisposition to Disease Caused by Phytophthora capsici

Pye, Matthew Francis University of California, Davis 2013 해외박사(DDOD)

소속기관이 구독 중이 아닌 경우 오후 4시부터 익일 오전 9시까지 원문보기가 가능합니다.

Plants respond to changes in the environment with complex signaling networks, often under the control of phytohormones that generate positive and negative crosstalk among downstream effectors of the response. Brief dehydration stresses such as salinity and water deficit, which induce a rapid and transient systemic increase in levels of abscisic acid (ABA), can influence disease response pathways. Plant defense responses to pathogens are mediated in part by the phytohormones salicylic acid (SA), jasmonic acid (JA) and ethylene (ET). ABA has been associated with susceptibility of plants to bacteria, fungi, and oomycetes but relatively little attention has been directed at its role in abiotic stress predisposition to root pathogens. Plant roots exposed to a brief episode of salt (sodium chloride) stress prior to infection are severely diseased relative to non-stressed plants. This study examines the impact of salinity stress on infection of tomato roots by Phytophthora capsici. An increase in root ABA levels in tomato preceded or temporally paralleled the onset of stress-induced susceptibility. ABA-deficient tomato mutants were shown to have a reduced predisposition response, which could be restored by complementation of the mutant with exogenous ABA. ABA related gene expression did not appear to be altered by P. capsici infection. Pathogenesis-related gene expression was induced in non-stressed plants during P. capsici infection. However, these genes were strongly suppressed in plants that had been salt-stressed prior to inoculation. SA- and JA-mediated responses in tomato roots are impacted by exposure to salinity as evidenced by the suppression of hallmark defense gene expression. Interference with SA or ET network functions does not significantly alter the predisposing effect of salt on disease severity. Rather, it appears that elevated ABA induced by salinity confers the dominant impact on the disease phenotype observed. This impact, which does not preclude effects on other phytohormone networks, is sufficient to predispose tomato to Phytophthora root and crown rot. In a second study, the plant activators Actigard (1,2,3-benzothiadiazole-7-thiocarboxylic acid-s-methyl-ester, BTH) and Tiadinil (N-(3-chloro-4-methylphenyl)-4- methyl-1,2,3-thiadiazole-5-carboxamide, TDL) were examined for their effects on disease and their impact on ABA-mediated, salinity-induced predisposition in hydroponically-grown tomato seedlings. An episode of salt stress to roots significantly increased the severity of disease caused by the bacterial speck pathogen Pseudomonas syringae pv. tomato (Pst) relative to non-stressed plants. In spite of the protection afforded by BTH and TDL to Pst, root treatment with these SAR activators increased the levels of ABA in roots and shoots similar to levels observed in salt-stressed plants. The results indicate that plant activators can protect tomato plants from bacterial speck disease under predisposing salt stress, and suggest that some SA-mediated defense responses may function sufficiently in plants with elevated levels of ABA.

Efficacy of Soybean Seed Treatments in Iowa and the Effect of Cold Stress on Damping-off Caused by Pythium sylvaticum

Serrano, Mauricio Iowa State University ProQuest Dissertations & The 2017 해외박사(DDOD)

소속기관이 구독 중이 아닌 경우 오후 4시부터 익일 오전 9시까지 원문보기가 가능합니다.

Soybean (Glycine max (L.) Merr.) production is negatively affected by the occurrence of seedling diseases. In Iowa, the occurrence of seedling diseases is commonly associated with cool and wet weather soon after planting, and several Pythium spp. have been found as the predominant pathogens causing seedling disease. Increasing cost of the seed, risk of seedling diseases reducing stand in cold, wet soils, and the yield benefit sometimes provided by early planting have motivated an increase in the use of fungicide seed treatment. However, a positive effect of seed treatments on soybean plant stand and yield has not been clearly demonstrated in Iowa. This study was conducted to: (i) compare the effect of commercially available soybean seed treatments on plant stand and yield; (ii) assess the effect of cold stress at planting on the incidence of Pythium seedling disease and efficacy of a commercial seed treatment; and (iii) evaluate the effect of chilling temperatures after planting on the Pythium--soybean interaction. Each year, three small plot field studies were conducted at different locations in Iowa in 2014, 2015 and 2016. Fifteen seed treatments were evaluated in 2014, and 13 seed treatments in 2015 and 2016. The experimental design was a randomized complete block with four replications. Seeding rate was 140.000 seeds per acre in 2014, and 120.000 seed per acre in 2015 and 2016. Periods of cool and wet weather soon after planting were observed in seven of nine trials within 4 to 7 days after planting. Enhancement of plant stand and yield by seed treatment was inconsistent among treatments and locations, even in trials where adverse weather conditions were observed soon after planting. Moreover, no bean leaf beetle (Cerotoma trifurcate) injury was observed, low soybean cyst nematode (SCN) (Heterodera glycines ) populations were measured at the trial sites, and sudden death syndrome (SDS) (Fusarium virguliforme) foliar disease index was in the trials was also low. Based on these findings, seed treatment may not be indispensable to achieve high plant stands in Iowa and should be regarded as a preventive control measure to protect soybean stand from seedling disease. Even though our results were inconclusive, as suggested by other authors farmers should consider the use of seed treatments in field has a history of SDS, SCN, or seedling disease particularly in early planting. The effect of cold stress on efficacy of a seed treatment containing metalaxyl and ethaboxam (Intego Suite(TM)) on soybean emergence was evaluated in a growth chamber experiment. A five-way factorial design with two cold stress temperatures (4°C and 10°C), two times for the initiation of cold stress (24 and 96 hours after planting), three cold stress durations (24, 48 and 96 hours), and two levels of seed treatments (Intego Suite(TM) and untreated) was conducted in cups inoculated with Pythium sylvaticum or a non-inoculated control. Emergence was reduced when the pathogen was present, and longer periods of cold stress further reduced emergence. No differences where observed between times for the initiation of cold stress. The cold stress had no effect on emergence when the pathogen was absent. Seed treatment protected soybean seedlings subjected to periods of cold stress. Seed treatment also resulted in improved shoot weight and reduced root rot severity. The results from this study demonstrated that seed treatment is a useful management tool to protect seedlings from damping-off caused by P. sylvaticum when soybeans are planted under suboptimal temperatures. To improve understanding of the effect of cold stress on Pythium damping-off, growth chamber and laboratory experiments were performed. A growth chamber study was conducted to test the effect of a 96-hour period of cold stress at different times after planting on soybean damping-off. The experimental design was a three way factorial with two cold stress temperatures (4°C and 10°C); seven levels of timing of the initiation of cold stress (no cold stress, 0, 1, 2, 4, 6, 8 days planting); and inoculation with P. sylvaticum-infested millet or sterile millet (non-inoculated control). Increased susceptibility to damping-off was observed when soybeans were subjected to cold stress. Emergence was assessed 21 days after planting and was particularly low when cold stress occurred 2 or 4 days after planting in inoculated cups. In the non-inoculated controls, no effect of cold stress on emergence was observed. Also, emergence and seedling growth was delayed when P. sylvaticum was present. In a laboratory experiment, mycelial growth of P. sylvaticum on diluted V8 media was assessed at different temperatures (4°C, 10°C and 18°C). Low temperatures delayed mycelial growth but the pathogen was still able to grow at 4°C. In another experiment, seed exudation was assessed by measuring electrical conductivity. Greater seed exudation was detected when soybean seeds were imbibed at 4°C compared to 10°C and 18°C. Sporangia of P. sylvaticum germinated in response to seed exudates, and sporangial germination increased when cultures were exposed to seed exudates from seeds imbibed at 4°C. These results demonstrate that the timing of cold stress during seed germination play an important role in the occurrence of damping-off caused by P. sylvaticum . Moreover, low temperatures increase seed exudation and may enhance activity of P. sylvaticum. The results from this study have improved our understanding of the soybean- Pythium interaction and conditions that favor disease development. These data will aid soybean farmers in Iowa in making their decisions whether or not to use a seed treatment.