Molecular characterisation of defence of Brassica napus (Oilseed rape) to Rhizoctonia solani AG2-1 confirmed by functional analysis in Arabidopsis thaliana

Rhizoctonia solani is a necrotrophic, soilborne fungal pathogen associated with significant establishment losses in Brassica napus (oilseed rape; OSR). The anastomosis group (AG) 2-1 of R. solani is the most virulent to OSR, causing damping-off, root and hypocotyl rot, and seedling death. Resistance to R. solani AG2-1 in OSR has not been identified, and the regulation of OSR defense to its adapted pathogen, AG2-1, has not been investigated. In this work, we used confocal microscopy to visualize the progress of infection by sclerotia of AG2-1 on B. napus varieties with contrasting disease phenotypes. We defined their defense response using gene expression studies and functional analysis with Arabidopsis thaliana mutants. Our results showed existing variation in susceptibility to AG2-1 and plant growth between OSR varieties, and differential expression of genes of hormonal and defense pathways related to auxin, ethylene, jasmonic acid, abscisic acid, salicylic acid, and reactive oxygen species regulation. Auxin, abscisic acid signaling, and the MYC2 branch of jasmonate signaling contributed to the susceptibility to AG2-1, while induced systemic resistance was enhanced by NAPDH RBOHD, ethylene signaling, and the ERF/PDF branch of jasmonate signaling. These results pave the way for future research, which will lead to the development of Brassica crops that are more resistant to AG2-1 of R. solani and reduce dependence on chemical control options

HAWAIIAN SKIRT, and F-box gene from Arabidopsis, is a new player in the microRNA pathway

F-box proteins belong to a multi-protein E3 ubiquitin ligase complex (SCF) that target proteins for degradation via the proteasome.We demonstrated that HAWAIIAN SKIRT(HWS), an Arabidopsis ubiquitin protein ligase (SCFHWS), regulates organ growth, flower development and timing of abscission. Mutants of this gene (hws-1) are pleiotropic and the most obvious phenotype is the fusion of its floral organs, a phenotype shared with the cuc1/cuc2 double mutants and over-expressing lines of MIR164B. To understand the molecular mechanisms of HWS during plant development, an ethylmethylsulphonate mutagenized population of hws-1 seeds was generated and screened for mutations suppressing the hws-1 sepal fusion. We isolated shs-1/hws-1, shs-2/hws-1, and shs-3/hws-1, (suppressor of hws-1) mutants. Mapping analyses shown that shs1 is mutated in the miRNA164 binding site of CUPSHAPED COTYLEDON1 (CUC1) mRNA; while shs-2 and shs-3 are novel alleles of the plant homolog of Exporting-5 HASTY (HST), known to be important in miRNA biogenesis, function and transport. Consequently, we renamed them cuc1-1D, hst23 and hst24, respectively. We demonstrated that transcript levels of CUC1 and CUPSHAPED COTYLEDON 2 (CUC2), and MIR164 change in cuc1-1D and in hws-1 mutants; analyses revealed a role for HWS in cell proliferation and control of floral organ number. Additional genetic crosses between hws-1 and mutant lines for genes in the miRNA pathway were performed and double mutants obtained shown restoration of the hws-1 sepal fusion phenotype. Our data propose HWS as a new regulator in miRNA pathway and reveal a role for HWS to control floral organ number and cell proliferation

Yield Losses and Control by Sedaxane and Fludioxonil of Soilborne Rhizoctonia, Microdochium, and Fusarium Species in Winter Wheat

Soilborne Rhizoctonia, Microdochium, and Fusarium species are major causal agents of seedling and stem-base diseases of wheat. Currently, seed treatments are considered the most effective solution for their control. Rhizoctonia solani anastomosis groups (AGs) 2-1 and 5, R. cerealis, Microdochium, and Fusarium spp., were used in series of field experiments to determine their capability to cause soilborne and stem-base disease and to quantify their comparative losses in the establishment and yield of wheat. The effectiveness and response to seed treatment formulated with 10 g sedaxane and 5 g fludioxonil 100 kg21 against these soilborne pathogens were also determined. Our results showed that damping-off caused by soilborne R. cerealis was associated with significant reductions in the emergence and establishment, resulting in stunted growth and low plant numbers. The pathogen also caused sharp eyespot associated with reductions in the ear partitioning index. R. solani AG 2-1 and AG 5 were weakly pathogenic and failed to cause significant damping-off, root rot, and stem-base disease in wheat. Fusarium graminearum and F. culmorum applied as soilborne inoculum failed to cause severe disease. Microdochium spp. caused brown foot rot disease and soilborne M. nivale reduced wheat emergence. Applications of sedaxane and fludioxonil increased plant emergence and reduced damping-off, early stem-base disease, and brown foot rot, thus providing protection against multiple soilborne pathogens. R. cerealis reduced the thousand grain weight by 3.6%, whereas seed treatment including fludioxonil and sedaxane against soilborne R. cerealis or M. nivale resulted in a 4% yield increase

Physiological, molecular, and genetic mechanism of action of the biostimulant Quantis™ for increased thermotolerance of potato (Solanum tuberosum L.)

Background: Raising global temperatures limit crop productivity and new strategies are needed to improve the resilience of thermosensitive crops such as potato (Solanum tuberosum L.). Biostimulants are emerging as potential crop protection products against environmental stress, however their mechanism of action remains largely unknown, hindering their wider adoption. We used comprehensive physiological, molecular, and mass spectrometry approaches to develop understanding of the mechanism of plant thermotolerance exerted by the biostimulant, Quantis™, under heat stress. Using orthologues gene mutations in Arabidopsis thaliana we report heat-defence genes, modified by Quantis™, which were also investigated for potential overlapping functions in biotic stress defence to Sclerotinia sclerotiorum and Rhizoctonia solani. Results: Quantis™ enhanced PSII photochemical efficiency and decreased thermal dissipation of potato grown under heat stress. These effects were associated with upregulation of genes with antioxidant function, including PR10, flavonoid 3′‐hydroxylase and β-glucosidases, and modulation of abscisic acid (ABA) and cytokinin (CK) activity in leaves by Quantis™. The biostimulant modulated the expression of the heat-defence genes, PEN1, PR4 or MEE59, with functions in leaf photoprotection and root thermal protection, but with no overlapping function in biotic stress defence. Protective root growth under heat stress, following the biostimulant application, was correlated with enhanced CK signalling in roots. Increased endogenous concentrations of ABA and CK in potato leaves and significant upregulation of StFKF1 were consistent with tuberisation promoting effects. Quantis™ application resulted in 4% tuber weight increase and 40% larger tuber size thus mitigating negative effects of heat stress on tuber growth. Conclusions: Quantis™ application prior to heat stress effectively primed heat tolerance responses and alleviated temperature stress of S. tuberosum L. and A. thaliana by modulating the expression and function of PR4 and MEE59 and by regulating CK activity above and below ground, indicating that the mechanism of action of the biostimulant is conserved, and will be effective in many plant species. Thus, a biostimulant application targeting the most susceptible crop developmental stages to heat disorders can be effectively integrated within future agronomy practices to mitigate losses in other thermosensitive crops

The role of photoprotection in defence of two wheat genotypes against Zymoseptoria tritici

This study provides new insights into the role of photoprotection in preformed and induced defence of two wheat genotypes with contrasting phenotypes to infection by Zymoseptoria tritici. We investigated the mechanisms of the photoprotective response during early infection, including nonphotochemical quenching (NPQ), β-carotene-derived xanthophylls, reactive oxygen species, and the phytohormones abscisic acid (ABA), jasmonic acid (JA), and salicylic acid (SA). Furthermore, we quantified the effects of pathogenesis on photosynthesis, stomatal control, and expression of plant defence molecular markers. The photoprotective mechanism of successful defence involved the qI component of NPQ leading to rapid down-regulation of photosystem II quantum yield and chlorophyll a:b, increased biosynthesis of the xanthophyll neoxanthin and ABA, and the expression of chloroplast-specific enzymes to engage in scavenging of O2●−. Elevated ABA in the resistant genotype correlated with preformed leaf defence traits including low stomatal density, increased expression of wax biosynthesis, and lignification. Z. tritici exhibited reduced germination and branching on the resistant host genotype and hijacked stomatal control in both genotypes by enhancing stomatal sensitivity to light. Increased biosynthesis of JA and anthocyanins, in contrast to SA, were quantified in the incompatible interaction. Our results indicate that ABA and JA in antagonistic action to SA were associated with defence in the resistant genotype, Cougar, against Z. tritici

The Arabidopsis thaliana F-box gene HAWAIIAN SKIRT is a new player in the microRNA pathway

In Arabidopsis, the F-box HAWAIIAN SKIRT (HWS) protein is important for organ growth. Loss of function of HWS exhibits pleiotropic phenotypes including sepal fusion. To dissect the HWS role, we EMS-mutagenized hws-1 seeds and screened for mutations that suppress hws-1 associated phenotypes. We identified shs-2 and shs-3 (suppressor of hws-2 and 3) mutants in which the sepal fusion phenotype of hws-1 was suppressed. shs-2 and shs-3 (renamed hst-23/hws-1 and hst-24/hws-1) carry transition mutations that result in premature terminations in the plant homolog of Exportin-5 HASTY (HST), known to be important in miRNA biogenesis, function and transport. Genetic crosses between hws-1 and mutant lines for genes in the miRNA pathway, also suppress the phenotypes associated with HWS loss of function, corroborating epistatic relations between the miRNA pathway genes and HWS. In agreement with these data, accumulation of miRNA is modified in HWS loss or gain of function mutants. Our data propose HWS as a new player in the miRNA pathway, important for plant growth

Dissecting the role of the Hawaiian Skirt gene in the regulation of floral development using suppressor analysis strategy

HAWAIIAN SKIRT (HWS) is an F-box gene in Arabidopsis that plays a key role in plant floral organ development. HWS has been identified due to sepal fusion along their basal margins resulting in failure to shed its floral organs (Gonzalez-Carranza el al., 2007). Similar phenotypic characteristics can be seen in the ectopically expressed microRNA miRl64 (Mallory et al., 2004; Lauf et al., 2004) and in the double mutants cup-shaped cotyledon 1 cuc1/Cuc2 (Aida el al., 1997). Previous studies carried out by Gonzalez-Carranza el al. (unpublished) using genetic crosses between hws-1 and other floral mutants has revealed that HWS may play a crucial role in the microRNA biogenesis. In an effort to identify potential substrates of HWS and to identity the role of HWS in the miRNA pathway, a population of EMS mutagenized hws•J was used for isolation and characterization of suppressors of hws-1. Screening a number of EMS mutagenized hws-1 populations has identified several suppressor lines that are currently under study. From the identified mutants, two lines 43 .1 and 80.5 were selected for further characterization analysis. These suppressor lines rescue the distinctive sepal fusion phenotype of hws1/ as well as displaying other phenotypic characteristics. Characterization of the suppressor lines has identified that 43.1/ is an allele of HST gene, which is involved in miRNA biogenesis, and 80.5 is an allele of AS2 gene, which is an adaxial cell fate determinant. Expression analyses have revealed that loss of HWS gene function leads to the repression of both 43./ and 80.5. Genetic analyses have also confirmed that loss of HWS gene function results in an upregulation of CUC] and CUC2 gene expression. The results obtained in this project have shown that HWS is involved in miRNA, adaxial-abaxial and Organ boundary signalling, concluding that HWS may have a wider function in different signalling pathways than previously proposed.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Yield loss and integrated disease control of Rhizoctonia solani AG2-1 using seed treatment and sowing rate of oilseed rape

Rhizoctonia solani anastomosis group (AG) 2-1 is an ubiquitous soil-borne pathogen causing severe damping-off of oilseed rape (OSR). In the absence of varietal resistance to AG2-1 there are limited methods for integrated disease management. The objectives of these field studies were to quantify yield losses due to AG2-1, and to determine the effectiveness of integrated control using sedaxane, fludioxonil and metalaxyl-M applied as seed treatment on two OSR genotypes at sowing rate of 40 (low) or 80 (high) seeds m-2. Crop assessments of green area index (GAI), vigor and cabbage stem flea beetle (CSFB) Psylliodes chrysocephala damage were carried out at GS16, whilst pathogen DNA in soil was quantified using real-time PCR at GS32. Yield and seed weight losses of 41% and 18%, respectively, were associated with reduced establishment, GAI, vigor, and delayed development and flowering of OSR. Seed treatment reduced AG2-1 DNA in soil by 80% resulting in a 94%, 16% and 64% increase of establishment, TSW and yield, respectively. Seed treatment also mitigated the effects of AG2-1 on delaying plant development resulting in increased uniformity of crop flowering. OSR plants infected with AG2-1 suffered 27% more damage by the CSFB indicating positive pathogen-pest interaction at the expense of the OSR host. Optimum control of AG2-1 infection was achieved by integrating low sowing rate and seed treatment. However, under dual pest and pathogen attack, high sowing rates should be combined with the use of seed treatment to mitigate seedling death and delayed development caused by AG2-1 and CSFB damage

HAWAIIAN SKIRT, and F-box gene from Arabidopsis, is a new player in the microRNA pathway

F-box proteins belong to a multi-protein E3 ubiquitin ligase complex (SCF) that target proteins for degradation via the proteasome.We demonstrated that HAWAIIAN SKIRT(HWS), an Arabidopsis ubiquitin protein ligase (SCFHWS), regulates organ growth, flower development and timing of abscission. Mutants of this gene (hws-1) are pleiotropic and the most obvious phenotype is the fusion of its floral organs, a phenotype shared with the cuc1/cuc2 double mutants and over-expressing lines of MIR164B. To understand the molecular mechanisms of HWS during plant development, an ethylmethylsulphonate mutagenized population of hws-1 seeds was generated and screened for mutations suppressing the hws-1 sepal fusion. We isolated shs-1/hws-1, shs-2/hws-1, and shs-3/hws-1, (suppressor of hws-1) mutants. Mapping analyses shown that shs1 is mutated in the miRNA164 binding site of CUPSHAPED COTYLEDON1 (CUC1) mRNA; while shs-2 and shs-3 are novel alleles of the plant homolog of Exporting-5 HASTY (HST), known to be important in miRNA biogenesis, function and transport. Consequently, we renamed them cuc1-1D, hst23 and hst24, respectively. We demonstrated that transcript levels of CUC1 and CUPSHAPED COTYLEDON 2 (CUC2), and MIR164 change in cuc1-1D and in hws-1 mutants; analyses revealed a role for HWS in cell proliferation and control of floral organ number. Additional genetic crosses between hws-1 and mutant lines for genes in the miRNA pathway were performed and double mutants obtained shown restoration of the hws-1 sepal fusion phenotype. Our data propose HWS as a new regulator in miRNA pathway and reveal a role for HWS to control floral organ number and cell proliferation

Mean of sepal and petal numbers in Col-0, <i>hws-1</i>, <i>hst-24/hws-1</i> and <i>hst-24</i> in Col-0 from the first 25 flowers of the inflorescences, (flowers n = 200).

<p>Mean of sepal and petal numbers in Col-0, <i>hws-1</i>, <i>hst-24/hws-1</i> and <i>hst-24</i> in Col-0 from the first 25 flowers of the inflorescences, (flowers n = 200).</p