The Arabidopsis mutant wrky33 is highly susceptible to Botrytis cinerea. We identified >1680 Botrytis-induced WRKY33 binding sites associated with 1576 Arabidopsis genes. Transcriptional profiling ...defined 318 functional direct target genes at 14 hr post inoculation. Comparative analyses revealed that WRKY33 possesses dual functionality acting either as a repressor or as an activator in a promoter-context dependent manner. We confirmed known WRKY33 targets involved in hormone signaling and phytoalexin biosynthesis, but also uncovered a novel negative role of abscisic acid (ABA) in resistance towards B. cinerea 2100. The ABA biosynthesis genes NCED3 and NCED5 were identified as direct targets required for WRKY33-mediated resistance. Loss-of-WRKY33 function resulted in elevated ABA levels and genetic studies confirmed that WRKY33 acts upstream of NCED3/NCED5 to negatively regulate ABA biosynthesis. This study provides the first detailed view of the genome-wide contribution of a specific plant transcription factor in modulating the transcriptional network associated with plant immunity.
Lipoxygenase (LOXs) is a large family of plant enzymes that catalyse the hydroperoxidation of free polyunsaturated fatty acids into diverse biologically active compounds, collectively named ...phyto-oxylipins. Although multiple isoforms of LOXs have been identified in a wide range of annual herbaceous plants, the genes encoding these enzymes in perennial woody plants have not received as much attention. In Camellia sinensis (L.) O. Kuntze, no LOX gene of any type has been isolated, and its possible role in tea plant development, senescence, and defence reaction remains unknown. The present study describes the isolation, characterization, and expression of the first tea plant LOX isoform, namely CsLOX1, and seeks to clarify the pattern of its expression in the plant's defence response as well as in flower opening and senescence.
Based on amino acid sequence similarity to plant LOXs, a LOX was identified in tea plant and named CsLOX1, which encodes a polypeptide comprising 861 amino acids and has a molecular mass of 97.8 kDa. Heterologous expression in yeast analysis showed that CsLOX1 protein conferred a dual positional specificity since it released both C-9 and C-13 oxidized products in equal proportion and hence was named 9/13-CsLOX1. The purified recombinant CsLOX1 protein exhibited optimum catalytic activity at pH 3.6 and 25°C. Real-time quantitative PCR analysis showed that CsLOX1 transcripts were detected predominantly in flowers, up-regulated during petal senescence, and down-regulated during flower bud opening. In leaves, the gene was up-regulated following injury or when treated with methyl jasmonate (MeJA), but salicylic acid (SA) did not induce such response. The gene was also rapidly and highly induced following feeding by the tea green leafhopper Empoasca vitis, whereas feeding by the tea aphid Toxoptera aurantii resulted in a pattern of alternating induction and suppression.
Analysis of the isolation and expression of the LOX gene in tea plant indicates that the acidic CsLOX1 together with its primary and end products plays an important role in regulating cell death related to flower senescence and the JA-related defensive reaction of the plant to phloem-feeders.
The large WRKY transcription factor family is mainly involved in regulating plant immune responses. Arabidopsis WRKY33 is a key transcriptional regulator of hormonal and metabolic processes towards ...Botrytis cinerea strain 2100 infection and is essential for resistance. In contrast to B. cinerea strain 2100, the strain B05.10 is virulent on wild‐type (WT) Col‐0 Arabidopsis plants highlighting the genetic diversity within this pathogen species. We analysed how early WRKY33‐dependent responses are affected upon infection with strain B05.10 and found that most of these responses were strongly dampened during this interaction. Ectopic expression of WRKY33 resulted in complete resistance towards this strain indicating that virulence of B05.10, at least partly, depends on suppressing WRKY33 expression/protein accumulation. As a consequence, the expression levels of direct WRKY33 target genes, including those involved in the biosynthesis of camalexin, were also reduced upon infection. Concomitantly, elevated levels of the phytohormone abscisic acid (ABA) were observed. Molecular and genetic studies revealed that ABA negatively influences defence to B05.10 and effects jasmonic acid/ethylene (JA/ET) and salicylic acid (SA) levels. Susceptibility/resistance was determined by the antagonistic effect of ABA on JA, and this crosstalk required suppressing WRKY33 functions at early infection stages. This indicates that B. cinerea B05.10 promotes disease by suppressing WRKY33‐mediated host defences.
Previously, we showed that resistance of Arabidopsis thaliana Col‐0 to Botrytis cinerea strain 2100 depends on the function of the transcription factor WRKY33, and this involves not only WRKY33‐dependent activation of genes encoding enzymes of camalexin biosynthesis but also WRKY33‐mediated negative regulation of ABA biosynthesis and ABA signalling. In the current study, we tested whether the susceptibility of Col‐0 plants towards the virulent B. cinerea strain B05.10 is due to suppression of host defences involving WRKY33. Our results provide molecular and genetic evidence that B. cinerea B05.10 infection dampens induced expression of WRKY33 at early fungal infection stages and thereby subsequently mitigates several WRKY33‐mediated host immune responses. In particular, this affects camalexin biosynthesis, and the antagonist affects ABA on JA signalling.
Tea, which is produced from new shoots of existing tea plants (
), is one of the most popular, non-alcoholic, healthy beverages worldwide.
is one of the dominant fungal pathogens of tea. The ...interaction of
with tea could be a useful pathosystem to elucidate various aspects of woody, medicinal plant-fungal interactions. Currently, many studies characterizing resistance or virulence and aggressiveness use lesion size at the infection sites on the leaves to quantify the growth of the pathogen. However, this method does not offer the sensitivity needed for the robust quantification of small changes in aggressiveness or the accurate quantification of pathogen growth at the early stages of infection.
A quantitative real-time polymerase chain reaction (qRT-PCR) assay was developed for the quantification of
growth on tea plant. This method was based on the comparison of fungal DNA in relation to plant biomass. This assay was used to investigate the phenotypes of tea plant cultivars in response to
infection. Two cultivars, Zhongcha 108 (ZC108) and Longjing 43 (LJ43), were tested with this method. ZC108 was previously reported as an anthracnose-resistant cultivar against
, while LJ43 was susceptible. The traditional lesion measurement method showed that both cultivars were susceptible to a virulent strain of
, while the qRT-PCR approach indicated that very little fungal growth occurred in the anthracnose-resistant cultivar ZC108. The observed results in this study were consistent with previously published research. In addition, the DNA-based real-time PCR method was applied for analysis of pathogenic differences in general
isolates and among several
spp that infect tea.
This study showed that the DNA-based qRT-PCR technique is rapid, highly sensitive and easily applicable for routine experiments and could be used in screening for resistant tea plant cultivars or to identify differences in pathogen aggressiveness within and among
species.
Meyer is one of the most valuable plants and is widely used in China, while ginseng anthracnose is one of the most destructive diseases.
could infect ginseng leaves and stems and causes serious ...anthracnose disease, but its mechanism is still unknown. Here, transcriptome and metabolism analyses of the host leaves were conducted to investigate the ginseng defense response affected by
. Upon
infection, ginseng transcripts altered from 14 to 24 h, and the expression of many defense-related genes switched from induction to repression. Consequently, ginseng metabolites in the flavonoid pathway were changed. Particularly,
repressed plant biosynthesis of the epicatechin and naringin while inducing plant biosynthesis of glycitin, vitexin/isovitexin, and luteolin-7-
-glucoside. This work indicates
successful infection of
by intervening in the transcripts of defense-related genes and manipulating the biosynthesis of secondary metabolites, which might have antifungal activities.
The tea plant
Camellia sinensis
(L.) O. Kuntze is one of the most important leaf crops, and it is widely used for the production of non-alcoholic beverages worldwide. Tea also has a long history of ...medicinal use.
Colletotrichum camelliae
Massee is one of the dominant fungal pathogens that infects tea leaves and causes severe tea anthracnose disease. To analyze the molecular biology of
C. camelliae
, the quantification of pathogen gene expression by the RT-qPCR method is necessary. Reliable RT-qPCR results require the use of stable reference genes for data normalization. However, suitable reference genes have not been reported in
C. camelliae
thus far. In this study, 12 candidate genes (i.e.,
CcSPAC6B12.04c
,
CcWDR83
,
Cchp11
,
Ccnew1
,
CcHplo
,
CcRNF5
,
CcHpcob
,
CcfaeB-2
,
CcYER010C
,
CcRNM1
,
CcUP18
, and
CcACT
) were isolated from
C. camelliae
and assessed as potential reference genes. The expression stability of these genes in
C. camelliae
during spore germination and mycelial growth and interaction with host plants was first evaluated using several statistical algorithms, such as geNorm, NormFinder, and Bestkeeper. A web-based analysis program, Refinder, was then used to find the most suitable reference genes. Our results indicated that
Cenew1
,
CcHplo
, and
CcSPAC6B12.04c
were the most stable reference genes in
C. camelliae
under all conditions. Our work provided the most suitable reference genes for future studies performed to quantify the target gene expression levels of
C. camelliae.
is a necrotrophic microbe that causes gray mold disease in a broad range of hosts. In the present study, we conducted molecular microbiology and transcriptomic analyses of the host-
interaction to ...investigate the plant defense response and fungal pathogenicity. Upon
infection, plant defense responses changed from activation to repression; thus, the expression of many defense genes decreased in
.
Zn(II)
Cys
transcription factor BcSpd1 was involved in the suppression of plant defense as Δ
altered wild-type B05.10 virulence by recovering part of the defense responses at the early infection stage. BcSpd1 affected genes involved in the fungal sclerotium development, infection cushion formation, biosynthesis of melanin, and change in environmental pH values, which were reported to influence fungal virulence. Specifically, BcSpd1 bound to the promoter of the gene encoding quercetin dioxygenase (
) and positively affected the gene expression, which was involved in catalyzing antifungal flavonoid degradation. This study indicates BcSpd1 plays a key role in the necrotrophic microbe
virulence toward plants by regulating pathogenicity-related compounds and thereby suppressing early plant defense.
Panax ginseng Meyer is one of the most valuable medicinal plants which is enriched in anti-microbe secondary metabolites and widely used in traditional medicine. Botrytis cinerea is a necrotrophic ...fungus that causes gray mold disease in a broad range of hosts. B. cinerea could overcome the ginseng defense and cause serious leaf and root diseases with unknown mechanism.
We conducted simultaneous transcriptomic and metabolomic analysis of the host to investigate the defense response of ginseng affected by B. cinerea. The gene deletion and replacement were then performed to study the pathogenic gene in B. cinerea during ginseng - fungi interaction.
Upon B. cinerea infection, ginseng defense responses were switched from the activation to repression, thus the expression of many defense genes decreased and the biosynthesis of antifungal metabolites were reduced. Particularly, ginseng metabolites like kaempferol, quercetin and luteolin which could inhibit fungi growth were decreased after B. cinerea infection. B. cinerea quercetin dioxygenase (Qdo) involved in catalyzing flavonoids degradation and △BcQdo mutants showed increased substrates accumulation and reduced disease development.
This work indicates the flavonoids play a role in ginseng defense and BcQdo involves in B. cinerea virulence towards the P. ginseng. B. cinerea promotes disease development in ginseng by suppressing of defense related genes expression and reduction of antifungal metabolites biosynthesis.
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Gray mold, caused by Botrytis cinerea, is one of the major fungal diseases in agriculture. Biological methods are preferred over chemical fungicides to control gray mold since they are less toxic to ...the environment and could induce the resistance to pathogens in plants. In this work, we try to understand if ginseng defense to B. cinerea could be induced by fungal hypovirulent strain △BcSpd1. BcSpd1 encodes Zn(II)2Cys6 transcription factor which regulates fungal pathogenicity and we recently reported △BcSpd1 mutants reduced fungal virulence.
We performed transcriptomic analysis of the host to investigate the induced defense response of ginseng treated by B. cinerea △BcSpd1. The metabolites in ginseng flavonoids pathway were determined by UPLC-ESI-MS/MS and the antifungal activates were then performed.
We found that △BcSpd1 enhanced the ginseng defense response when applied to healthy ginseng leaves and further changed the metabolism of flavonoids. Compared with untreated plants, the application of △BcSpd1 on ginseng leaves significantly increased the accumulation of p-coumaric acid and myricetin, which could inhibit the fungal growth.
B. cinerea △BcSpd1 could effectively induce the medicinal plant defense and is referred to as the biological control agent in ginseng disease management.
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Tea is one of the most well-known, healthy beverages in the world. Tea plants produce caffeine as a secondary metabolite.
is one of the most important microbes frequently isolated from tea fields, ...and it causes anthracnose disease in tea plant. In the present work, we performed molecular microbiology and transcriptomic analyses of the
- tea plant interaction to investigate the mechanism of fungal virulence and plant defense. Upon infection of tea plant with
, we observed alterations in the expression of fungal transcripts, including those of many genes associated with caffeine metabolism, such as those encoding various transporters, xanthine dehydrogenase, and urate oxidase (UOX). In particular, the deletion of
urate oxidase (
), which is involved in the caffeine metabolism pathway, reduced fungal tolerance to caffeine, and impaired fungal virulence. CcUOX is involved in caffeine metabolism by the degradation of uric acid contents.
Δ
mutants impaired uric acid degradation
. The
gene was cloned from
, overexpressed in
, and the recombinant CcUOX protein displayed maximum activity at 30°C and a pH of 4.0. The recombinant CcUOX efficiently reduced uric acid
suggesting a promising application in caffeine-contaminated environment management and in producing food with low purine contents to prevent uric acid related human diseases, such as hyperuricemia and gout.
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