Biochemical characterisation of class III biotin protein ligases from Botrytis cinerea and Zymoseptoria tritici

Pectin is a major cell wall component that plays important roles in plant development and response to environmental stresses. Arabidopsis thaliana plants expressing a fungal polygalacturonase (PG plants) that degrades homogalacturonan (HG), a major pectin component, as well as loss-of-function mutants for QUASIMODO2 (QUA2), encoding a putative pectin methyltransferase important for HG biosynthesis, show accumulation of reactive oxygen species (ROS), reduced growth and almost complete resistance to the fungal pathogen Botrytis cinerea. Both PG and qua2 plants show increased expression of the class III peroxidase AtPRX71 that contributes to their elevated ROS levels and reduced growth. In this work, we show that leaves of PG and qua2 plants display greatly increased cuticle permeability. Both increased cuticle permeability and resistance to B. cinerea in qua2 are suppressed by loss of AtPRX71. Increased cuticle permeability in qua2, rather than on defects in cuticle ultrastructure or cutin composition, appears to be dependent on reduced epidermal cell adhesion, which is exacerbated by AtPRX71, and is suppressed by the esmeralda1 mutation, which also reverts the adhesion defect and the resistant phenotype. Increased cuticle permeability, accumulation of ROS, and resistance to B. cinerea are also observed in mutants lacking a functional FERONIA, a receptor-like kinase thought to monitor pectin integrity. In contrast, mutants with defects in other structural components of primary cell wall do not have a defective cuticle and are normally susceptible to the fungus. Our results suggest that disrupted cuticle integrity, mediated by peroxidase-dependent ROS accumulation, plays a major role in the robust resistance to B. cinerea of plants with altered HG integrity.

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Characterization of Iprodione Resistance in Botrytis cinerea from Strawberry and Blackberry

Phytopathology ◽

10.1094/phyto-06-13-0156-r ◽

2014 ◽

Vol 104 (4) ◽

pp. 396-402 ◽

Cited By ~ 47

Author(s):

Anja Grabke ◽

Dolores Fernández-Ortuño ◽

Achour Amiri ◽

Xingpeng Li ◽

Natália A. Peres ◽

...

Keyword(s):

North Carolina ◽

South Carolina ◽

Botrytis Cinerea ◽

Efflux Pump ◽

Point Mutations ◽

Convincing Evidence ◽

Class Iii ◽

Multidrug Efflux Pump ◽

Single Spore ◽

Pump Activity

Gray mold, caused by the fungal pathogen Botrytis cinerea, is one of the most destructive diseases of strawberry. Control of the disease in commercial fields is largely dependent on the application of fungicides, including the dicarboximide iprodione. Single-spore isolates were collected from strawberry fields in Florida, North Carolina, and South Carolina and subjected to an assay using conidial germination that distinguished sensitive (S) isolates from isolates with various levels of resistance to iprodione. Of the 245 isolates, 1 was highly resistant (HR), 5 were moderately resistant (MR), and 43 had low resistance (LR) to iprodione. LR and MR strains were found in the Florida population and in 9 of 11 locations from North Carolina and South Carolina, indicating that resistance was widespread but accounted for only a relatively small percentage of the B. cinerea population. Sequence analysis of the target gene bos1, which codes for a class III histidine kinase, revealed that the MR phenotype was associated with Q369P and N373S mutations and that the LR phenotype was associated with either a I365S or a I365N mutation. The I365S and I365N mutations were also present in five additionally included HR isolates from North Carolina and South Carolina blackberry fields and one HR isolate from a Virginia strawberry field but no mutation or mutation combinations in bos1 were uniquely associated with the HR phenotype. Expression analysis of bos1 in S and HR isolates did not reveal convincing evidence of the gene's involvement in HR resistance either. The six HR isolates had three different phenotypes with respect to their sensitivity to fludioxonil; two were S, two were LR, and two were MR. The fludioxonil LR and MR isolates were also resistant to tolnaftate, an indication of multidrug efflux pump activity. These data suggest that, in addition to point mutations in bos1, drug efflux pump activity and potentially a third mechanism of resistance may be contributing to the iprodione HR phenotype. Detached fruit studies showed that field rates of Rovral 4 Flowable (iprodione) did not control iprodione MR and HR isolates.

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A Class III Histidine Kinase Acts as a Novel Virulence Factor in Botrytis cinerea

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi-19-1042 ◽

2006 ◽

Vol 19 (9) ◽

pp. 1042-1050 ◽

Cited By ~ 109

Author(s):

Muriel Viaud ◽

Sabine Fillinger ◽

Weiwei Liu ◽

Jai Santosh Polepalli ◽

Pascal Le Pêcheur ◽

...

Keyword(s):

Botrytis Cinerea ◽

Culture Media ◽

Class Iii ◽

In Planta ◽

Gene Encoding ◽

Group Iii ◽

Oxidative Stresses ◽

Large Numbers ◽

Standard Culture ◽

Null Mutants

Filamentous ascomycetes contain large numbers of histidine kinases (HK) that belong to eleven classes. Members of class III from different species were previously shown to be involved in osmoregulation and resistance to dicarboximide and phenylpyrrole fungicides. We have inactivated the gene encoding the single group III HK, BOS1, in the economically important plant pathogen Botrytis cinerea. BOS1 inactivation had pleiotropic effects on the fungus. Besides the expected osmosensitivity and resistance to fungicides, null mutants presented additional characteristics indicating that BOS1 is necessary for normal macroconidiation and full virulence. On standard culture media, null mutants very rarely formed conidiophores and those few conidiophores failed to produce conidia. This defect could be partially restored with 1 M sorbitol, suggesting that another BOS1-independent signal cascade may be involved in macroconidiation. The mutants were not found to be hypersensitive to various oxidative stresses but were more resistant to menadione. Finally, pathogenicity tests showed that bos1-null mutants were significantly reduced in the ability to infect host plants. Appressorium morphogenesis was not altered; however, in planta growth was severely reduced. To our knowledge, this is the first class III HK characterized as a pathogenicity factor in a plant-pathogenic ascomycete.

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First Report of Fenpyrazamine Resistance in Botrytis cinerea from Strawberry Fields in Spain

Plant Health Progress ◽

10.1094/php-12-17-0075-br ◽

2018 ◽

Vol 19 (1) ◽

pp. 45-45

Author(s):

Dolores Fernández-Ortuño ◽

Alejandra Vielba-Fernández ◽

Alejandro Pérez-García ◽

Juan A. Torés ◽

Antonio de Vicente

Keyword(s):

Botrytis Cinerea ◽

Gray Mold ◽

In Vivo Studies ◽

Cross Resistance ◽

In Vitro Tests ◽

Class Iii ◽

Single Spore ◽

First Report

Botrytis cinerea Pers. is an important fungal pathogen responsible for gray mold, one of the most economically important diseases of strawberry (Fragaria × ananassa) worldwide. The primary disease management strategy involves the application of different classes of fungicides, including the sterol biosynthesis inhibitor class III fungicide fenpyrazamine. In 2014 and 2015, strawberries affected with gray mold symptoms were collected from eight locations in Huelva, where fenhexamid had been used extensively. Twenty-five B. cinerea single-spore isolates were examined to determine EC50 values and to determine a discriminatory dose to monitor fenpyrazamine resistance in the field in future studies. The in vitro tests divided the isolates into two groups: 15 sensitive (EC50 from 0.02 to 1.3 μg/ml) and 10 resistant (EC50 from 50.1 to 172.6 μg/ml), which showed cross-resistance with fenhexamid. Performance of fenpyrazamine in in vivo studies was also carried out. Only the fenpyrazamine-resistant isolates developed gray mold on the fungicide-treated fruit. This is the first report of fenpyrazamine resistance in B. cinerea from strawberry fields in Spain and cross-resistance with fenhexamid.

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Disruption of the Bcchs3a Chitin Synthase Gene in Botrytis cinerea Is Responsible for Altered Adhesion and Overstimulation of Host Plant Immunity

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi-02-10-0046 ◽

2010 ◽

Vol 23 (10) ◽

pp. 1324-1334 ◽

Cited By ~ 20

Author(s):

Delphine Arbelet ◽

Pierrette Malfatti ◽

Elizabeth Simond-Côte ◽

Thierry Fontaine ◽

Loïc Desquilbet ◽

...

Keyword(s):

Cell Wall ◽

Botrytis Cinerea ◽

Critical Role ◽

Infection Process ◽

Chitin Synthase ◽

Phytopathogenic Fungus ◽

Class Iii ◽

Fungal Cell ◽

Fungal Virulence ◽

Isolation And Characterization

The fungal cell wall is a dynamic structure that protects the cell from different environmental stresses suggesting that wall synthesizing enzymes are of great importance for fungal virulence. Previously, we reported the isolation and characterization of a mutant in class III chitin synthase, Bcchs3a, in the phytopathogenic fungus Botrytis cinerea. We demonstrated that virulence of this mutant is severely impaired. Here, we describe the virulence phenotype of the cell-wall mutant Bcchs3a on the model plant Arabidopsis thaliana and analyze its virulence properties, using a variety of A. thaliana mutants. We found that mutant Bcchs3a is virulent on pad2 and pad3 mutant leaves defective in camalexin. Mutant Bcchs3a was not more susceptible towards camalexin than the wild-type strain but induced phytoalexin accumulation at the infection site on Col-0 plants. Moreover, this increase in camalexin was correlated with overexpression of the PAD3 gene observed as early as 18 h postinoculation. The infection process of the mutant mycelium was always delayed by 48 h, even on pad3 plants, probably because of lack of mycelium adhesion. No loss in virulence was found when Bcchs3a conidia were used as the inoculum source. Collectively, these data led us to assign a critical role to the BcCHS3a chitin synthase isoform, both in fungal virulence and plant defense response.

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