Ion currents associated with root tips, emerging laterals and induced wound sites in Nicotians tabacum: spatial relationship proposed between resulting electrical fields and phytophthoran zoospore infection

Abstract Growing roots of Nicotiana tabacum var. Havana generate transcellular ion currents which traverse developing and wounded tissues. Positive current of around 10 mA m^?2 enters meristematic and elongating cells at the tip of primary roots. The growing tips of first order laterals are also traversed by a similar positive current with a density of around 2.0 mA m^?2, as are immature laterals emerging at the primary root surface. These self-generated ion currents flow basipetally through developing tissues and leave from mature non-elongating tissue. A large positive current of around 70 mA m^?2 also enters induced wound sites on the primary root surface. Motile zoospores of the fungal pathogen Phytophthora parasitica var. nicotianae have been reported to associate preferentially with these regions of the root. This might suggest that electrotaxis may be part of the mechanism by which zoospores locate root regions susceptable to fungal infection.     

Two Phytophthora parasitica cysteine protease genes,PpCys44 and PpCys45, trigger cell death in various Nicotiana spp. and act as virulence factors

Proteases secreted by pathogens have been shown to be important virulence factors modifying plant immunity, and cysteine proteases have been demonstrated to participate in different pathosystems. However, the virulence functions of the cysteine proteases secreted by Phytophthora parasitica are poorly understood. Using a publicly available genome database, we identified 80 cysteine proteases in P. parasitica, 21 of which were shown to be secreted. Most of the secreted cysteine proteases are conserved among different P. parasitica strains and are induced during infection. The secreted cysteine protease proteins PpCys44/45 (proteases with identical protein sequences) and PpCys69 triggered cell death on the leaves of different Nicotiana spp. A truncated mutant of PpCys44/45 lacking a signal peptide failed to trigger cell death, suggesting that PpCys44/45 functions in the apoplastic space. Analysis of three catalytic site mutants showed that the enzyme activity of PpCys44/45 is required for its ability to trigger cell death. A virus-induced gene silencing assay showed that PpCys44/45 does not induce cell death on NPK1 (Nicotiana Protein Kinase 1)-silenced Nicotiana benthamiana plants, indicating that the cell death phenotype triggered by PpCys44/45 is dependent on NPK1. PpCys44- and PpCys45-deficient double mutants showed decreased virulence, suggesting that PpCys44 and PpCys45 positively promote pathogen virulence during infection. PpCys44 and PpCys45 are important virulence factors of P. parasitica and trigger NPK1-dependent cell death in various Nicotiana spp.     

In vitro and in vivo antimicrobial efficacy of essential oils and individual compounds against Phytophthora parasitica var. nicotianae

Aim

To evaluate the antimicrobial effects of essential oils (EOs) from cassia, basil, geranium, lemongrass, cumin and thyme, as well as their major components, against Phytophthora parasitica var. nicotianae; to investigate morphological changes in hyphae and sporangia in response to treatment with cinnamaldehyde; and to further evaluate potential biocontrol capacities against tobacco black shank under greenhouse conditions.

Methods and Results

The results revealed that the extent of mycelial growth inhibition was primarily dependent on the composition and concentration of the EOs and the structure of individual compounds. Cinnamaldehyde had a significantly higher inhibitory effect on mycelial growth, formation of sporangia, and production and germination of zoospores in P. parasitica var. nicotianae in vitro, achieving complete inhibition of these phenotypes at 72, 36, 36 and 18 mg l^?1, respectively. Scanning electron microscopic observations revealed that cinnamaldehyde can cause considerable morphological degenerations of hyphae and sporangia such as cytoplasmic coagulation, shrivelled mycelia and sporangia aggregates and swelling and lysis of mycelia and sporangia walls. In vivo assays with cinnamaldehyde demonstrated that this compound afforded protective effect against tobacco black shank under greenhouse conditions in susceptible tobacco plants.

Conclusions

The results of in vitro and in vivo bioassays, together with SEM imaging of the microstructure of P. parasitica var. nicotianae supported the possibility of using cinnamaldehyde as a potent natural biofungicide in the greenhouse.

Significance and Impact of the Study

This study provides a theoretical basis for the potential use of cinnamaldehyde as commercial agents or lead compounds that can be exploited as commercial biofungicides in the protection of tobacco plants from P. parasitica var. nicotianae infection.     

The Raf-like kinase Raf36 negatively regulates plant resistance against the oomycete pathogen Phytophthora parasitica by targeting MKK2

Oomycetes represent a unique group of plant pathogens that are phylogenetically distant from true fungi and cause significant crop losses and environmental damage. Understanding of the genetic basis of host plant susceptibility facilitates the development of novel disease resistance strategies. In this study, we report the identification of an Arabidopsis thaliana T-DNA mutant with enhanced resistance to Phytophthora parasitica with an insertion in the Raf-like mitogen-activated protein kinase kinase kinase gene Raf36. We generated additional raf36 mutants by CRISPR/Cas9 technology as well as Raf36 complementation and overexpression transformants, with consistent results of infection assays showing that Raf36 mediates Arabidopsis susceptibility to P. parasitica. Using a virus-induced gene silencing assay, we silenced Raf36 homologous genes in Nicotiana benthamiana and demonstrated by infection assays the conserved immune function of Raf36. Mutagenesis analyses indicated that the kinase activity of Raf36 is important for its immune function and interaction with MKK2, a MAPK kinase. By generating and analysing mkk2 mutants and MKK2 complementation and overexpression transformants, we found that MKK2 is a positive immune regulator in the response to P. parasitica infection. Furthermore, infection assay on mkk2 raf36 double mutant plants indicated that MKK2 is required for the raf36-conferred resistance to P. parasitica. Taken together, we identified a Raf-like kinase Raf36 as a novel plant susceptibility factor that functions upstream of MKK2 and directly targets it to negatively regulate plant resistance to P. parasitica.     

Study of mRNA Expression by Real Time PCR of Cpkk1, Cpkk2 and Cpkk3, three MEKs of Cryphonectria parasitica,in Virus‐free and Virus‐infected Isogenic Isolates

Cpkk1 and Cpkk2 are two previously characterized Mitogen‐activated protein kinase kinases (MEK) from Cryphonectria parasitica. For the characterization of the third MEK, primers designed to a conserved region of the known fungal MEK sequences were used in a PCR reaction to amplify genomic DNA from C. parasitica. The sequence of the resulting amplicon was compared to known sequences in the database using a Blast search. Results of the sequence comparison indicated that the initial fragment obtained encoded for a new MEK from C. parasitica, that had highest homology to Pbs2 from Saccharomyces cerevisiae. By inverse PCR we obtained a genomic fragment spanning the entire coding sequence of this MEK, which was named Cpkk3. The cDNA of Cpkk3 was obtained by compiling the sequences of RT‐PCR products resulting from the amplification of purified mRNA. TaqMan^® Probes were designed to analyse the expression of Cpkk1, Cpkk2 and Cpkk3 mRNA through RT‐Real Time PCR. This protocol allowed the expression of Cpkk3 to be successfully compared to the expression of Cpkk1 and Cpkk2, two previously cloned C. parasitica MEKs. No variation in expression was associated with the presence of a virus after 2 days of growth in standard conditions whereas an increase in the expression level of all the three MEKs was shown after 4 days of growth.     

Pullulans produced by strains of Cryphonectria parasitica—II. Nuclear magnetic resonance evidence

The structure of pullullan-like polysaccharides produced as exocellular material by different strains of Cryphonectria parasitica, the fungus responsible for chestnut tree cankers, was investigated with nuclear magnetic resonance (NMR) techniques. ¹³C, mono- and bidimensional ¹H, and ¹H–¹³C heteronuclear correlated NMR spectra (HSQC and HMBC) were recorded. Advanced analysis of the NMR spectra allowed the main resonance of the atoms in the maltotriose and in the maltotetraose repeat units of pullulan-like polysaccharides from C. parasitica to be recognised with confidence. In all cases investigated, the presence of large amounts of -(1→6) maltotetraose subunits was evidenced, in addition to the -(1→6) maltotriose subunits, corresponding to the repeating unit of pullulan produced by Aureobasidium pullulans and other fungi. The results were in agreement with other data from this laboratory, obtained with independent techniques. The belief that in ‘pullulans’ the maximum amount of -(1→6) maltotetraose subunits is about 7% can thus be considered as definitely outdated.     

Tête à tête inside a plant cell: establishing compatibility between plants and biotrophic fungi and oomycetes

'Compatibility' describes the complementary relationship between a plant species and an adapted pathogen species that underlies susceptibility and which ultimately results in disease. Owing to elaborate surveillance systems and defence mechanisms on the plant side and a common lack of adaptation of many microbial pathogens, resistance is the rule and compatibility the exception for most plant-microbe combinations. While there has been major scientific interest in 'resistance' in the past decade, which has revealed many of its underlying molecular components, the analysis of 'compatibility', although intimately intertwined with 'resistance', has not been pursued with a similar intensity. Various recent studies, however, provide a first glimpse of the pivotal players and potential molecular mechanisms essential for compatibility in both the plant and parasite partners. In this review we highlight these findings with a particular emphasis on obligate biotrophic and hemibiotrophic fungal and oomycete pathogens and discuss novel strategies that might help to uncover further the molecular principles underlying compatibility to these highly specialized pathogens.     

The chestnut blight fungus world tour: successive introduction events from diverse origins in an invasive plant fungal pathogen

Clonal expansion has been observed in several invasive fungal plant pathogens colonizing new areas, raising the question of the origin of clonal lineages. Using microsatellite markers, we retraced the evolutionary history of introduction of the chestnut blight fungus, Cryphonectria parasitica, in North America and western Europe. Combining discriminant analysis of principal components and approximate Bayesian computation analysis, we showed that several introduction events from genetically differentiated source populations have occurred in both invaded areas. In addition, a low signal of genetic recombination among different source populations was suggested in North America. Finally, two genetic lineages were present in both invaded areas as well as in the native areas, suggesting the existence of genetic lineages with a high capacity to establish in diverse environments and host species. This study confirmed the importance of multiple introductions, but questioned the role of genetic admixture in the success of introduction of a fungal plant pathogen.