NBS-LRR Proteins and Their Partners:Molecular Switches of Plant Defense

Specificity of the plant innate immune system is often conferred by resistance（R）proteins.Most plant disease resistance （R）proteins contain a series of leucine-rich repeats（LRRs）,a nucleotide-binding site（NBS）,and a putative amino-terminal signaling domain.They are termed NBS-LRR proteins.The LRRs are mainly involved in recognition,and the amino-terminal domain determines signaling specificity,whereas the NBS domain presumably functions as a molecular switch.During the past years,the most important discoveries are the role of partners in NBS-LRR gene mediated defenses,mounting support for the so-called＂guard hypothesis＂of R gene function,and providing evidence for intramolecular interactions and intermolecular interactions within NBS- LRR proteins as a mode of signaling regulation.The outcome of these interactions determines whether a plant activates its defense responses.

Advances in the Role of DNA Methylation and miRNAs in Plant Defense

DNA methylation and miRNAs are two important epigenetic molecular mechmxisms in plants. RNA-directed DNA methylation （RdDM） pathway is con- sidered as the most important mechanism of DNA methylation. A microRNA （miRNA） is processed from a 70-90-nucleotide-long single-stranded RNA precursor that forms a hairpin structure by Dicer enzyme, which plays an important role in plant development, regulation of plant gene expression and silencing of trans- posons. In recent years, a large number of studies have confirmed that DNA methylation and miRNAs play an important role in disease resistance mechanisms of plants. In this paper, recent progress on the role of DNA methylation and miRNAs in plant defense was summarized, which provided reference for revealing the re- lationship between disease resistance factors and epigenctic mechanisms in plants.

Phase separation of the nuclear pore complex facilitates selective nuclear transport to regulate plant defense against pathogen and pest invasion

The nuclear pore complex(NPC),the sole exchange channel between the nucleus and cytoplasm,is composed of several subcomplexes,among which the central barrier determines the permeability/selectivity of the NPC to dominate the nucleocytoplasmic trafficking essential for many important signaling events in yeast and mammals.How plant NPC central barrier controls selective transport is a crucial question remaining to be elucidated.In this study,we uncovered that phase separation of the central barrier is critical for the permeability and selectivity of plant NPC in the regulation of various biotic stresses.Phenotypic assays of nup62 mutants and complementary lines showed that NUP62 positively regulates plant defense against Botrytis cinerea,one of the world’s most disastrous plant pathogens.Furthermore,in vivo imaging and in vitro biochemical evidence revealed that plant NPC central barrier undergoes phase separation to regulate selective nucleocytoplasmic transport of immune regulators,as exemplified by MPK3,essential for plant resistance to B.cinerea.Moreover,genetic analysis demonstrated that NPC phase separation plays an important role in plant defense against fungal and bacterial infection as well as insect attack.These findings reveal that phase separation of the NPC central barrier serves as an important mechanism to mediate nucleocytoplasmic transport of immune regulators and activate plant defense against a broad range of biotic stresses.

Quantitative disease resistance:Multifaceted players in plant defense

In contrast to large-effect qualitative disease resistance,quantitative disease resistance(QDR)exhibits partial and generally durable resistance and has been extensively utilized in crop breeding.The molecular mechanisms underlying QDR remain largely unknown but considerable progress has been made in this area in recent years.In this review,we summarize the genes that have been associated with plant QDR and their biological functions.Many QDR genes belong to the canonical resistance gene categories with predicted functions in pathogen perception,signal transduction,phytohormone homeostasis,metabolite transport and biosynthesis,and epigenetic regulation.However,other"atypical"QDR genes are predicted to be involved in processes that are not commonly associated with disease resistance,such as vesicle trafficking,molecular chaperones,and others.This diversity of function for QDR genes contrasts with qualitative resistance,which is often based on the actions of nucleotidebinding leucine-rich repeat(NLR)resistance proteins.An understanding of the diversity of QDR mechanisms and of which mechanisms are effective against which classes of pathogens will enable the more effective deployment of QDR to produce more durably resistant,resilient crops.

Indirect plant defense against insect herbivores： a review

Plants respond to herbivore attack by launching 2 types of defenses： direct defense and indirect defense. Direct defense includes all plant traits that increase the resistance of host plants to insect herbivores by affecting the physiology and/or be- havior of the attackers. Indirect defense includes all traits that by themselves do not have significant direct impact on the attacking herbivores, but can attract natural en- emies of the herbivores and thus reduce plant loss. When plants recognize herbivore- associated elicitors, they produce and release a blend of volatiles that can attract preda- tors, parasites, and other natural enemies. Known herbivore-associated elicitors include fatty acid-amino acid conjugates, sulfur-containing fatty acids, fragments of cell walls, peptides, esters, and enzymes. Identified plant volatiles include terpenes, nitrogenous compounds, and indoles. In addition, constitive traits including extrafloral nectars, food bodies, and domatia can be further induced to higher levels and attract natural enemies as well as provide food and shelter to carnivores. A better understanding of indirect plant defense at global and componential levels via advanced high throughput technolo- gies may lead to utilization of indirect defense in suppression of herbivore damage to plants.

Salicylic acid-mediated plant defense： Recent developments, missing links, and future outlook

BACKGROUND： Plant pathogens are responsible for many of history＇s greatest famines. Understanding how plants defend themselves against pathogens is crucial to preventing future famines. Salicylic acid （SA）-mediated plant defense is a key defense pathway, which plants use to defend against biotrophic and hemi-biotrophic pathogens. As a master regulator of SA- mediated plant defense, NPR1 interacts with TGA and WRKY transcription factor families, individual members of which positively or negatively regulate plant defense. OBJECTIVE： In this review we describe the recent developments and predict future directions of research on the involvement of circadian rhythm-, autophagy-, and viral RNA silencing-related genes in SA-mediated plant defense on SA, on plant defense, the induction effects of PR proteins, and the mechanisms by which NPR1 regulates defense-related genes. METHODS： We performed an extensive search of current and past literature using the PubMed, Google Scholar, and Google search engines. Our search terms included： ＂SA-mediated plant defense,＂ and ＂NPR1 [AND] salicylic acid.＂ Other search terms, wildcards, and Boolean operators were paired with ＂NPRI＂ or ＂plant defense＂ as needed to research more detailed information related to specific topics covered within this review. We also used Google to search for, ＂economic impact citrus greening,＂ ＂aspirin,＂ ＂Irish potato famine,＂ and ＂rice blast,＂ among other terms, to gather background information on the history and impact of plant diseases, and the historical use of aspirin. RESULTS： Of 148 sources found, 132 were directly related to plant defense. The remaining sources are related to the historical and economic impact of plant diseases and the historical use and mechanism of action of aspirin or salicylate. All reviewed sources have been documented in the references section. CONCLUSION： The topic of salicylic acid-mediated plant defense is broad, and new research is expanding our understanding of this topic quickly. In this review, we give a basic overview of the historical economic impact of plant diseases, and how an understanding of SA-mediated plant defense can prevent future famines. We provide a basic overview of plant defense, then discuss how SA acts as a defense signaling molecule.We discuss how SA regulates NPR1, which goes on to activate expression of SA-related genes including PR genes. Later, we discuss current research topics, including the role of NPR1 and SA in autophagy, circadian rhythmicity, viral gene silencing, SA biosynthesis, and SAR. We also discuss the potential roles of PR proteins, other SA binding proteins, WRKY and TGA family transcription factors, Elongator, and ER transport proteins in plant defense. Finally, we discuss the potential future routes that research into this topic could take, in order to further our understanding of role SA plays in plant defense.

Gene Expression:A Review on Methods for the Study of Defense-Related Gene Differential Expression in Plants

The plant genes involved in cellular signaling and metabolism have not been fully identified, while the function(s) of many of those which have are as yet incompletely characterized. Gene expression analysis allows the identification of genes and the study of their relationship with cellular processes. There are several options available for studying gene expression, including the use of cDNA and microarray libraries and techniques such as suppression subtractive hybridization (SSH), differential display (DD), RNA fingerprinting by arbitrary primed PCR (RAP), expressed sequence tags (EST), serial analysis of gene expression (SAGE), representational difference analysis (RDA), cDNA-amplified fragment length polymorphism (cDNA-AFLP) and RNA sequencing (RNA-Seq). Focusing on defense-related processes in plants, we present a brief review and examples of each of these methodologies and their advantages and limitations regarding the study of plant gene expression.

A chorismate mutase from Radopholus similis plays an essential role in pathogenicity

In the process of infecting plants, plant parasitic nematodes release a series of proteins that play an essential role in the successful infection and pathogenesis of plant cells and tissues through stylets or body walls. In this study,based on transcriptome data, a chorismate mutase gene of Radopholus similis(RsCM) was identified and cloned,which is a single copy gene specifically expressed in the oesophageal gland and highly expressed in juveniles and females. Transient expression of RsCM in tobacco leaves showed that it was localised in the cytoplasm and nucleus of tobacco leaf cells, which inhibited the pattern-triggered immunity(PTI) induced by flg22, including callose deposition and defence gene expression, and cell death induced by immune elicitors BAX, but could not inhibit cell death induced by immune elicitors Gpa2/RBP-1. The RNA interference(RNAi) transgenic tomato of RsCM obviously inhibited the infection, pathogenicity, and reproduction of R. similis. However, the resistance of the overexpression transgenic tomato of RsCM to R. similis infection was significantly reduced, and the expression levels of two salicylic acid(SA) pathway genes(PR1 and PR5) in roots infected by the nematode were significantly down-regulated,which indicated that RsCM might be involved in the inhibition of SA pathway. The results of this study demonstrate that RsCM suppresses the host immune system and might be a new target for the control of R. similis, which also provides new data for the function and mechanism of CM genes of migratory parasitic plant nematodes.

Ozone disrupts the communication between plants and insects in urban and suburban areas:an updated insight on plant volatiles

Plant-insect interactions are basic components of biodiversity conservation.To attain the international Sustainable Development Goals(SDGs),the interactions in urban and in suburban systems should be better understood to maintain the health of green infrastructure.The role of ground-level ozone(0_(3)) as an environmental stress disrupting interaction webs is presented.Ozone mixing ratios in suburbs are usually higher than in the center of cities and may reduce photosynthetic productivity at a relatively higher degree.Consequently,carbon-based defense capacities of plants may be suppressed by elevated 0_(3) more in the suburbs.However,contrary to this expectation,grazing damages by leaf beetles have been severe in some urban centers in comparison with the suburbs.To explain differences in grazing damages between urban areas and suburbs,the disruption of atmospheric communication signals by elevated 0_(3) via changes in plant-regulated biogenic volatile organic compounds and long-chain fatty acids are considered.The ecological roles of plant volatiles and the effects of 0_(3) from both a chemical and a biological perspective are presented.Ozone-disrupted plant volatiles should be considered to explain herbivory phenomena in urban and suburban systems.

Evaluation of cotton germplasm for morphological and biochemical host plant UPdates resistance traits against sucking insect pests complex

Background:Sucking insect pests cause severe damage to cotton crop production.The development of insect resistant cotton cultivars is one of the most effective measures in curtailing the yield losses.Considering the role of morphological and biochemical host plant resista nee(HPR)traits in plant defense,12 cotton genotypes/varieties were evaluated for leaf area,leaf glanding,total soluble sugars,total soluble proteins,total phenolics,tannin and total flavonoids against fluctuating populations of whitefly,thrips and jassid under field conditions.Results:The population of these insects fluctuated during the growing seas on and remained above threshold level(whitefly>5,thrips>(8-10)f or jassid>1 per leaf)during late June and early July.Strong and negative association of whitefly(r=-0.825)and jassid(r=-0.929)with seed cotton yield was observed.Mean population of insects were the highest in Glandless-1 followed by NIA-82 and NIA-M30.NIAB-Kiran followed by NI AB-878 and Sadori were the most resistant,with the mean population of 1.41,1.60,1.66(whitefly);2.24,232,2.53(thrips)and 037,0.31,036(jassid),respectively.The resistant variety NIAB-Kiran showed less soluble sugars(8.54 mg.g^(-1)),soluble proteins(27.11 mg.g^(-1))and more phenolic(36.56 mg.g^(-1))and flavonoids(13.10mg.g^(-1))as compared with the susceptible check Glandless-1.Moreover,all insect populations were positively correlated with total soluble sugars and proteins.Whitefly populations exhibited negative response to leaf gossypol glands,total phenolics,tannins and flavonoids.The thrips and jassid populations had a significant and negative correlation with these four biochemical HPR traits.Conclusion:The ide ntified resistant resources and HPR traits can be deployed against sucking in sect pests'complex in future breeding programs of developing insect resistant cotton varieties.

m^(6)A modification of plant virus enables host recognition by NMD factors in plants

N^(6)-methyladenosine(m^(6)A)is the most abundant eukaryotic mRNA modification and is involved in various biological processes.Increasing evidence has implicated that m^(6)Amodification is an important anti-viral defense mechanism in mammals and plants,but it is largely unknown how m^(6)Aregulates viral infection in plants.Here we report the dynamic changes and functional anatomy of m^(6)Ain Nicotiana benthamiana and Solanum lycopersicum during Pepino mosaic virus(PepMV)infection.m^(6)Amodification in the PepMV RNA genome is conserved in these two species.Overexpression of the m^(6)Awriters,mRNA adenosine methylase A(MTA),and HAKAI inhibit the PepMV RNA accumulation accompanied by increased viral m^(6)Amodifications,whereas deficiency of these writers decreases the viral RNA m^(6)Alevels but enhances virus infection.Further study reveals that the cytoplasmic YTH-domain family protein NbECT2A/2B/2C as m^(6)Areaders are involved in anti-viral immunity.Protein-protein interactions indicate that NbECT2A/2B/2C interact with nonsense-mediated mRNA decay(NMD)-related proteins,including NbUPF3 and NbSMG7,but not with NbUPF1.m^(6)Amodification-mediated restriction to PepMV infection is dependent on NMD-related factors.These findings provide new insights into the functionality of m^(6)Aanti-viral activity and reveal a distinct immune response that NMD factors recognize the m^(6)Areaders-viral m^(6)ARNA complex for viral RNA degradation to limit virus infection in plants.

Biochemical Mechanism Unlocking Their Potential Role in Salt Tolerance Mechanism of Zizyphus Germplasm

Salinity is one of the major constraints reducing plant growth and yield.Irrigation with poor quality and brackish water to orchards is a major cause of stunted growth and low yield.The salt tolerance mechanism is one of the complicated genomic characters that is very problematic to develop in fruit trees and becomes much more severe at any growth and developmental stage.Osmotic stress and hormonal imbalances are major constraints causing low biomass production.Fruit tree tolerance/sensitivity is chiefly based on the activation of a defense system comprised of super-oxidase dismutase(SOD),peroxidase(POD)and catalases(CAT),non-enzymatic compounds including ascorbic acid,phenolics,flavonoids,stress indicators[i.e.,hydrogen peroxide(H2O2),lipid peroxidation,malondialdehyde(MDA),reactive oxygen species(ROS)and osmolytes containing proline,glycine-betaine(GB),ascorbates(APX),glutathione peroxidase(GPX)and glutathione reductase(GR)].Tolerant genotypes must have higher antioxidant assays to cope with the adverse effects of salinity stress because their defense system had the potential to scavenge toxic ROS and protect from membrane leakage.Some work is conducted on agronomic and horticultural crops;however,underutilized fruit crops are still neglected and need serious consideration from plant researchers.Minor fruit crops especially Zizyphus had excellent nutritional aspects.The current study provides detailed insights into the physiological and biochemical mechanisms of Zizyphus species to cope with the adverse effects of salinity by improving their plant defense system.The development of salt-tolerant germplasm is a requisite and can be developed by utilization of physiological,biochemical,and molecular mechanisms.Application of different molecular approaches(i.e.,genome mapping,genome editing,genetic transformation,proteomics,transcriptomics,and metabolites)are effective for higher yield by improving tolerance mechanisms.

Transcriptome and metabolome analysis reveal that oral secretions from Helicoverpa armigera and Spodoptera litura influence wound-induced host response in cotton

Cotton(Gossypium hirsutum)is an important fiber crop worldwide.Insect attack causes cotton yield and quality losses.However,little is known about the mechanism of cotton response to insect attack.We simulated insect feeding by applying insect oral secretions(OS)to wounds,and combined transcriptome and metabolome analysis to investigate how OS from two major pest species(Helicoverpa armigera and Spodoptera litura)affect cotton defense responses.We found that respectively 12,668 and 13,379 genes were differentially expressed in comparison with wounding alone.On addition of OS,the jasmonic acid signaling pathway was rapidly and strongly induced,whereas genes involved in salicylic acid biosynthesis were downregulated.On constructing a coexpression gene network,we identified a hub gene encoding a leucine-rich repeat receptor kinase that may play an important role in early signal recognition and transduction.OS from the two insect species altered the abundance of flavonoid-related compounds in different patterns.Gossypol remained in lower concentration after OS application than after wounding alone,suggesting a suppressive effect of OS on cotton defense response.This study illustrated transcriptional and metabolic changes of cotton in responding to OS from two chewing insect species,identified potential key response genes,and revealed evidence for OS inhibition of wounding-induced cotton defense response.

Effect of strawberry vein banding virus and strawberry mottle virus co-infection on the growth and development of strawberry

Strawberry mottle virus(SMoV)and strawberry vein banding virus(SVBV)cause diseases on strawberry plants,but the effect of coinfection of SMoV and SVBV on the growth,development,and defense system of strawberry(Fragaria×ananassa Duchesne)remains unknown.We investigated the effect of SMoV and SVBV coinfection on strawberry cultivar‘Benihope’.The results showed that stem diameter,leaf size,leaf number,relative chlorophyll content,total chlorophyll content,photosynthetic parameters,and stomatal aperture of SMoV and SVBV co-infected strawberry(VIS)plants were in a weaker level than uninfected control plants,indicating that viruses inhibited the growth and photosynthesis of strawberry plants.Furthermore,the initiation of flowering and fruiting stages of VIS plants were delayed by about three weeks compared with the controls,and the fruiting period was shortened,demonstrating that the reproduction of VIS plants was inhibited.Fruit quality was damaged in VIS plants due to a significant increase in fruit firmness and titratable acidity and decrease in total soluble solid content than control fruits.More dead cells and H_(2)O_(2) accumulated along the veins of VIS leaves,and the content of abscisic acid and catalase activity significantly increased,whereas anthocyanin content was lower than that of control plants.The results demonstrate that SVBV and SMoV coinfection inhibits the growth and development of‘Benihope’strawberry plants,and the plants respond to viruses by regulating stomatal aperture,the accumulation of ABA and antioxidants.To our knowledge,this study contributes information to understand how both viruses impair the strawberry growth and development for the first time.

Promotion of Arabidopsis immune responses by a rhizosphere fungus via supply of pipecolic acid to plants and selective augment of phytoalexins

The ascomycete insect pathogenic fungi such as Metarhizium species have been demonstrated with the abilities to form the rhizosphere or endophytic relationships with different plants for nutrient exchanges.In this study,after the evident infeasibility of bacterial disease development in the boxed sterile soils,we established a hydroponic system for the gnotobiotic growth of Arabidopsis thaliana with the wild-type and transgenic strain of Metarhizium robertsii.The transgenic fungus could produce a high amount of pipecolic acid(PIP),a pivotal plant-immune-stimulating metabolite.Fungal inoculation experiments showed that M.robertsii could form a non-selective rhizosphere relationship with Arabidopsis.Similar to the PIP uptake by plants after exogenous application,PIP level increased in Col-0 and could be detected in the PIP-non-producing Arabidopsis mutant(ald1)after fungal inoculations,indicating that plants can absorb the PIP produced by fungi.The transgenic fungal strain had a better efficacy than the wild type to defend plants against the bacterial pathogen and aphid attacks.Contrary to ald1,fmo1 plants could not be boosted to resist bacterial infection after treatments.After fungal inoculations,the phytoalexins camalexin and aliphatic glucosinolate were selectively increased in Arabidopsis via both PIP-dependent and-independent ways.This study unveils the potential mechanism of the fungus-mediated beneficial promotion of plant immunity against biological stresses.The data also highlight the added values of M.robertsii to plants beyond the direct suppression of insect pest populations.

Global Change Effects on Plant Chemical Defenses against Insect Herbivores

This review focuses on individual effects of major global change factors, such as elevated CO2, O3, UV light and temperature, on plant secondary chemistry. These secondary metabolites are well-known for their role in plant defense against insect herbivory. Global change effects on secondary chemicals appear to be plant species-specifc and dependent on the chemical type. Even though plant chemical responses induced by these factors are highly variable, there seems to be some specificity in the response to different environmental stressors. For example, even though the production of phenolic compounds is enhanced by both elevated CO2 and UV light levels, the latter appears to primarily increase the concentrations of flavonoids. Likewise, specific phenolic metabolites seem to be induced by O3 but not by other factors, and an increase in volatile organic compounds has been particularly detected under elevated temperature. More information is needed regarding how global change factors influence inducibility of plant chemical defenses as well as how their indirect and direct effects impact insect performance and behavior, herbivory rates and pathogen attack. This knowledge is crucial to better understand how plants and their associated natural enemies will be affected in future changing environments.

Influences of the Populus deltoids seedlings treated with exogenous methyl jasmonate on the growth and development of Lymantria dispar larvae

Lyantria dispar larvae were fed with the leaves of Populus deltoids seedlings exposed to methyl jasmonate （MeJA） for 24 h. The growth and development of the larvae were investigated, and phenolics contents in treated leaves including pyrocatechol, caffeic acid, coumarin, fernlic acid and benzoic acid were also surveyed by high-pressure liquid chromatography （HPLC）. Results indicated that approximate digestibility, efficiency of conversion of ingested food, efficiency of conversion of digested food, and weight of the larvae were inhibited obviously, especially from the sixth day, which may result from the increase of total phenolics contents in treated leaves. This result provides strong supports for MeJA acting as the airborne signal molecule between woody plants.

Salicylic Acid and its Function in Plant Immunity

The small phenolic compound salicylic acid （SA） plays an important regulatory role in multiple physiological processes including plant im- mune response. Significant progress has been made during the past two decades in understanding the SA-mediated defense signaling network. Characterization of a number of genes functioning in SA biosynthesis, conjugation, accumulation, signaling, and crosstalk with other hormones such as jasmonic acid, ethylene, abscisic acid, auxin, gibberellic acid, cytokinin, brassinosteroid, and peptide hormones has sketched the finely tuned immune response network. Full understanding of the mech- anism of plant immunity will need to take advantage of fast developing genomics tools and bioinformatics techniques. However, elucidating genetic components involved in these pathways by conventional ge- netics, biochemistry, and molecular biology approaches will continue to be a major task of the community. High-throughput method for SA quantification holds the potential for isolating additional mutants related to SA-mediated defense signaling.

Plant pleiotropic drug resistance transporters:Transport mechanism, gene expression,and function

Pleiotropic drug resistance （PDR） transporters belonging to the ABCG subfamily of ATP-binding cassette （ABC） transporters are identified only in fungi and plants. Members of this family are expressed in plants in response to various biotic and abiotic stresses and transport a diverse array of moleculesacross membranes, Although their detailed transport mechanism is largely unknown, they play important roles in detoxification processes, preventing water loss, transport of phytohormones, and secondary metabolites. This review provides insights into transport mechanisms of plant PDR transporters, their expression profiles, and multitude functions in plants.

Jasmonate- and salicylate-induced defenses in wheat affect host preference and probing behavior but not performance of the grain aphid, Sitobion avenae

Jasmonate and salicylatemediated signaling pathways play significant roles in induced plant defenses, but there is no sufficient evidence for their roles in monocots against aphids. We exogenously applied methyl jasmonate （MeJA） and salicylic acid （SA） on wheat seedlings and examined biochemical responses in wheat and effects on the grain aphid, Sitobion avenae （Fab.）. Application of MeJA significantly increased levels of wheat＇s polyphenol oxidase, peroxidase and proteinase inhibitor 1, 2 and 6 days after treatment. In twochoice tests, adult aphids preferred control wheat leaves to MeJA or SA treated leaves. Electrical penetration graph （EPG） recordings of aphid probing behavior revealed that on MeJAtreated plants, the duration of aphid＇s first probe was significantly shorter and number of probes was significantly higher than those on control plants. Also total duration of probing on MeJAtreated plants was significantly shorter than on control plants. Total duration of salivation period on SAtreated plants was significantly longer, while mean phloem ingestion period was significantly shorter than on control plants. However, no significant difference in total duration of phloem sap ingestion period was observed among treatments. The EPG data suggest that MeJAdependent resistance factors might be due to feeding deterrents in mesophyll, whereas the SAmediated resistance may be phloembased. We did not observe any significant difference of MeJA and SA application on aphid development, daily fecundity, intrinsic growth rate and population growth. The results indicate that both MeJA and SAinduced defenses in wheat deterred S. avenae colonization processes and feeding behavior, but had no significant effects on its performance.