Biosynthesis of hydroxyphaseollin and related isoflavanoids in disease-resistant soybean hypocotyls

Phenylalanine-U-¹⁴C and isoliquiritigenin-9-¹⁴C were readily incorporated into the antifungal pterocarpan hydroxyphaseollin in soybean hypocotyls that were inoculated with incompatible strains of the phytopathogenic fungus Phytophthora megasperma var. sojae. Hydroxyphaseollin accounted for over half of the phenylalanine and isoliquiritigenin incorporated into ethyl acetate soluble compounds. Daidzein, coumestrol, and sojagol were identified as major compounds which accumulated coordinately with hydroxyphaseollin and contained significant amounts of radioactivity from the labelled isoflavanoid precursors. Hydroxyphaseollin was not present in healthy soybean plants and was not detected until ca. 16 hr after inoculation with the fungus. The pterocarpan then accumulated rapidly between 16 and 48 hr after inoculation, while the greatest accumulations of daidzein, coumestrol, and sojagol occurred between 48 and 72 hr after inoculation, when hydroxyphaseollin accumulation had ceased. Although soybean hypocotyls contained the anthocyanin malvin, neither this compound nor any other flavone pathway product was observed to accumulate after fungus-inoculation. The results therefore indicate that the accumulation of hydroxyphaseollin in fungus-inoculated soybean hypocotyls involves the activation of isoflavanoid biosynthesis with 'direction' of metabolic intermediates to biosynthesis of the pterocarpan.     

Aerobiology in plant protection

Summary The control of phytopathogenic fungi in agriculture requires sometimes excessive chemical treatments because, in many cases, reliable monitoring systems in order to assess the real risk for the cultivations are not available.Therefore, the likelihood to assess the presence and the amount of phytophatogenic fungus propagules in the air with a spore-trap has been investigated in order to evaluate their correlation with the climate and the disease occurrence. Four phytopathogenic fungi, namelyVenturia inequalis, Phytophthora infestans, Cercospora beticola andDiaporthe phaseolorum var.caulivora were taken into consideration.Practical use of airborne spore monitoring in a disease control strategy proved to be useful only forVenturia inequalis andPhytophthora infestans causal agent of apple scab and tomato late blight respectively. For the latter, the utilisation of an aerobiological monitoring system for the disease prediction can be assumed.     

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.     

Rerouting of Plant Late Endocytic Trafficking Toward a Pathogen Interface

A number of plant pathogenic and symbiotic microbes produce specialized cellular structures that invade host cells where they remain enveloped by host‐derived membranes. The mechanisms underlying the biogenesis and functions of host–microbe interfaces are poorly understood. Here, we show that plant late endocytic trafficking is diverted toward the extrahaustorial membrane (EHM); a host–pathogen interface that develops in plant cells invaded by Irish potato famine pathogen Phytophthora infestans. A late endosome and tonoplast marker protein Rab7 GTPase RabG3c, but not a tonoplast‐localized sucrose transporter, is recruited to the EHM, suggesting specific rerouting of vacuole‐targeted late endosomes to a host–pathogen interface. We revealed the dynamic nature of this process by showing that, upon activation, a cell surface immune receptor traffics toward the haustorial interface. Our work provides insight into the biogenesis of the EHM and reveals dynamic processes that recruit membrane compartments and immune receptors to this host–pathogen interface.      

Proteomic profile of the plant‐pathogenic oomycete Phytophthora capsici in response to the fungicide pyrimorph

Pyrimorph is a novel fungicide from the carboxylic acid amide (CAA) family used to control plant‐pathogenic oomycetes such as Phytophthora capsici. The proteomic response of P. capsici to pyrimorph was investigated using the iTRAQ technology to determine the target site of the fungicide and potential biomarker candidates of drug efficacy. A total of 1336 unique proteins were identified from the mycelium of wild‐type P. capsici isolate (Hd3) and two pyrimorph‐resistant mutants (R3‐1 and R3‐2) grown in the presence or absence of pyrimorph. Comparative analysis revealed that the three P. capsici isolates Hd3, R3‐1, and R3‐2 produced 163, 77, and 13 unique proteins, respectively, which exhibited altered levels of abundance in response to the pyrimorph treatment. Further investigations, using Cluster of Orthologous Groups of Proteins (COG) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis identified 35 proteins related to the mode of action of pyrimorph against P. capsici and 62 proteins involved in the stress response of P. capsici to pyrimorph. Many of the proteins with altered expression were associated with glucose and energy metabolism. Biochemical analysis using d ‐[U‐¹⁴C]glucose verified the proteomics data, suggesting that the major mode of action of pyrimorph in P. capsici is the inhibition of cell wall biosynthesis. These results also illustrate that proteomics approaches are useful tools for determining the pathways targeted by novel fungicides as well as for evaluating the tolerance of plant pathogens to environmental challenges, such as the presence of fungicides.