RESUMEN
Background: RAPID ALKALINIZATION FACTOR (RALFs) are cysteine-rich peptides that regulate multiple physiological processes in plants. This peptide family has considerably expanded during land plant evolution, but the role of ancient RALFs in modulating stress responses is unknown.Results: Here, we used the moss Physcomitrium patens as a model to gain insight into the role of RALF peptides in the coordination of plant growth and stress response in non-vascular plants. The quantitative proteomic analysis revealed concerted downregulation of M6 metalloprotease and some membrane proteins, including those involved in stress response, in PpRALF1, 2 and 3 knockout (KO) lines. The subsequent analysis revealed the role of PpRALF3 in growth regulation under abiotic and biotic stress conditions, implying the importance of RALFs in responding to various adverse conditions in bryophytes. We found that knockout of the PpRALF2 and PpRALF3 genes resulted in increased resistance to bacterial and fungal phytopathogens, Pectobacterium carotovorum and Fusarium solani, suggesting the role of these peptides in negative regulation of the immune response in P. patens. Comparing the transcriptomes of PpRALF3 KO and wild-type plants infected by F. solani showed that the regulation of genes in the phenylpropanoid pathway and those involved in cell wall modification and biogenesis was different in these two genotypes. Conclusion: Thus, our study sheds light on the function of the previously uncharacterized PpRALF3 peptide and gives a clue to the ancestral functions of RALF peptides in plant stress response.
RESUMEN
A new species, Trichocladium solani, was isolated from potato (Solanum tuberosum L.) tubers from Russia. The species has no observed teleomorph and is characterized morphologically by non-specific Acremonium-like conidia on single phialides and chains of swollen chlamydospores. Phylogenetic analysis placed the new species in a monophyletic clade inside the Trichocladium lineage with a high level of support from a multi-locus analysis of three gene regions: ITS, tub2, and rpb2. ITS is found to be insufficient for species delimitation and is not recommended for identification purposes in screening studies. T. solani is pathogenic to potato tubers and causes lesions that look similar to symptoms of Fusarium dry rot infection but with yellowish or greenish tint in the necrotized area. The disease has been named "yellow rot of potato tubers".
RESUMEN
Early blight of potato (Solanum tuberosum) is caused by Alternaria species and occurs annually in major potato producing regions of Russia. Diseased potato leaves displaying early blight symptoms were collected in July 2016 from a commercial field in Primorsky Krai, Russia (43.8242° N, 131.6219° E). The disease incidence was 30 to 40%. The initial symptoms appeared as typical early blight symptoms with a dark brown margin and diffused chlorosis on the leaf blade. Symptomatic leaves from different plants were randomly collected to isolate axenic cultures of the causal agents. Infected leaves were placed in wet chambers (moist filter papers in Petri dishes), and incubated at 25°C, 16 h/8 h dark/light photoperiod for 2-4 d. Single conidia were transferred to potato dextrose agar (PDA, Crous et al. 2009) in Petri dishes and incubated at 25°C for 7 d in the dark. Colonies were white-olivaceous, reverse side - olivaceous. Isolates were transferred onto potato carrot agar (PCA, Crous et al. 2009) and incubated at 22°C under a 16 h/8 h dark/light photoperiod for 7 d to stimulate sporulation. Most isolates (85%) were identified as A. protenta according to the morphological characteristics and molecular data, although one isolate showed sporulation that was somewhat atypical, having a smaller (especially narrower or more slender) conidia. Conidiophores were long, erect, and 65 to 100 µm × 5 to 6 µm in size. Conidia were solitary, long-ovoid in body with six to eight transverse septa, and 85 to 100 µm× 6 to 10 µm in size. Conidial beaks were filamentous, 110 to 200 µm × 2 to 5 µm in size. Genomic DNA was extracted from cultured isolates using the CTAB-chloroform extraction method (Griffith & Shaw 1998), and five gene regions including the internal transcribed spacer (ITS) region, translation elongation factor 1-alpha (tef1), RNA polymerase second largest subunit (rpb2), Alternaria major allergen (Alt a 1) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) genes were amplified with the primer pairs ITS1/ITS4, EF1-728F/EF1-986R, RPB2-5F2/FRPB2-7cR, Alt-for/Alt-rev, and gpd1/gpd2 respectively (Woudenberg et al. 2014). PCR products were Sanger sequenced. All sequences for isolate A16PrPL21 were identical to isolate CBS 105.51; (accession nos.: ITS, KJ718105; tef1, KJ718454; rpb2, KJ718279; Alt a 1, KJ718625; gpd KJ717959) of A. alternariacida Woudenb. & Crous. ITS, tef1, rpb2, Alt a 1 and gpd sequences were deposited in GenBank under the accessions OM348531, MN580518, MN580529, MN562258 and MN544404 respectively. Based on morphological and molecular data, the isolate was identified as Alternaria alternariacida Woudenb. & Crous. A. alternariacida is closely related to A. silybi, which is also found in the Far East (Woudenberg et al. 2014). Phylogenetic distances between these strains are based on differences at the ITS, TEF1 and Alt a 1 gene regions. A pathogenicity test was carried out with isolate A16PrPL21 on nine 5-week-old healthy potato plants (cv. Nevsky) grown in a greenhouse at 23 ± 2 °C. Seed tubers were grown in the greenhouse to obtain the seedlings. Inoculation was performed by spraying a conidial suspension (105 spores/ml) prepared from 10-day-old A. alternariacida culture grown on PCA at 23° C with a 12-h photoperiod. Nine negative control plants were treated with sterile distilled water. The inoculated plants were then maintained in a greenhouse at 25 °C with high humidity and 12-h light period. All test plants were covered with plastic bags for 24 h to maintain high relative humidity and incubated at 24 to 28°C. Leaf spot symptoms, brown lesions with chlorotic halos, similar to those previously observed in naturally infected plants, appeared 5 d post inoculation for all inoculated plants. After 7 d, the spots reached 18 to 25 mm in size. The symptoms were similar to the original symptoms that occurred in the field. Negative control leaves were symptomless. Koch's postulates were fulfilled by reisolating the pathogen from inoculated leaves and identified as A. alternaricida by rpb2 gene sequence and morphological characteristics. To our knowledge, this is the first report of disease caused by A. alternaricida on potato plants. Early blight, caused by large-spored Alternaria, is a widespread disease on potato. A. alternaricida is among a group of species that cause early blight, according to current research. Studies of the Alternaria species' biology and analyses of their distribution are important for improving potato protection from early blight.
RESUMEN
BACKGROUND: Cryptic peptides (cryptides) are small bioactive molecules generated via degradation of functionally active proteins. Only a few examples of plant cryptides playing an important role in plant defense have been reported to date, hence our knowledge about cryptic signals hidden in protein structure remains very limited. Moreover, little is known about how stress conditions influence the size of endogenous peptide pools, and which of these peptides themselves have biological functions is currently unclear. RESULTS: Here, we used mass spectrometry to comprehensively analyze the endogenous peptide pools generated from functionally active proteins inside the cell and in the secretome from the model plant Physcomitrella patens. Overall, we identified approximately 4,000 intracellular and approximately 500 secreted peptides. We found that the secretome and cellular peptidomes did not show significant overlap and that respective protein precursors have very different protein degradation patterns. We showed that treatment with the plant stress hormone methyl jasmonate induced specific proteolysis of new functional proteins and the release of bioactive peptides having an antimicrobial activity and capable to elicit the expression of plant defense genes. Finally, we showed that the inhibition of protease activity during methyl jasmonate treatment decreased the secretome antimicrobial potential, suggesting an important role of peptides released from proteins in immune response. CONCLUSIONS: Using mass-spectrometry, in vitro experiments and bioinformatics analysis, we found that methyl jasmonate acid induces significant changes in the peptide pools and that some of the resulting peptides possess antimicrobial and regulatory activities. Moreover, our study provides a list of peptides for further study of potential plant cryptides.