Overexpression of Solanum tuberosum Respiratory Burst Oxidase Homolog A (StRbohA) Promotes Potato Tolerance to Phytophthora infestans.

Reactive oxygen species (ROSs) represent one of the first lines of plants' biochemical defense against pathogens. Plants' respiratory burst oxidase homologs (RBOHs) produce ROSs as byproducts in several cellular compartments. In potato tubers, Solanum tuberosum respiratory burst oxidase homolog (StRBOHs) are involved in suberization and healing of wounded tissues. StRbohA has been tested in the model plant Arabidopsis thaliana, which led to enhanced plant defense against the soilborne pathogen Verticillium dahliae. Here, we showed that overexpressing StRbohA in potato plants increases plant tolerance to the oomycete Phytophthora infestans, the causal agent of late blight disease. Transgenic potato plants expressing StRbohA showed reduced disease symptoms (necrosis) compared with the wild type. In parallel, the expression of pathogenesis-related genes (PRs); RBOHs; antioxidation-related genes CPRX1, PRX2, APRX1, CAT1, and CAT2; and genes involved in the biosynthesis pathways of jasmonic and salicylic acids (ICS, PAL1, PAL2, LOX1, LOX2, and LOX3) exhibited significant increases in transgenic plants in response to infection. After higher expression of RBOHs, ROSs accumulated more in inoculation sites of the transgenic plants. ROSs act as signals that activate gene expression in the salicylic acid (SA) biosynthesis pathway, leading to the accumulation of SA and triggering SA-based defense mechanisms. SA-responsive PRs showed higher expression in the transgenic plants, which resulted in the restriction of pathogen growth in plant tissues. These results demonstrate the effective role of StRbohA in increasing potato defense against P. infestans.

Goss's Wilt Resistance in Corn Is Mediated via Salicylic Acid and Programmed Cell Death but Not Jasmonic Acid Pathways.

A highly aggressive strain (CMN14-5-1) of Clavibacter nebraskensis bacteria, which causes Goss's wilt in corn, induced severe symptoms in a susceptible corn line (CO447), resulting in water-soaked lesions followed by necrosis within a few days. A tolerant line (CO450) inoculated with the same strain exhibited only mild symptoms such as chlorosis, freckling, and necrosis that did not progress after the first six days following infection. Both lesion length and disease severity were measured using the area under the disease progression curve (AUDPC), and significant differences were found between treatments. We analyzed the expression of key genes related to plant defense in both corn lines challenged with the CMN14-5-1 strain. Allene oxide synthase (ZmAOS), a gene responsible for the production of jasmonic acid (JA), was induced in the CO447 line in response to CMN14-5-1. Following inoculation with CMN14-5-1, the CO450 line demonstrated a higher expression of salicylic acid (SA)-related genes, ZmPAL and ZmPR-1, compared to the CO447 line. In the CO450 line, four genes related to programmed cell death (PCD) were upregulated: respiratory burst oxidase homolog protein D (ZmrbohD), polyphenol oxidase (ZmPPO1), ras-related protein 7 (ZmRab7), and peptidyl-prolyl cis-trans isomerase (ZmPPI). The differential gene expression in response to CMN14-5-1 between the two corn lines provided an indication that SA and PCD are involved in the regulation of corn defense responses against Goss's wilt disease, whereas JA may be contributing to disease susceptibility.

Applications of CPPs in Genome Editing of Plants.

Cell penetrating peptides (CPPs) are short peptides that are able to translocate themselves and their cargo into cells. The progressive and continuous application of CPPs in various fields of basic and applied research shows that they are efficient delivery vectors for an assortment of biomolecules, including nucleic acids and proteins. This feature makes CPPs an excellent tool for modification of plant genomes through transgenesis and genome editing. In this review, we present the progress during the last three decades in application of CPPs for delivery of DNA, RNA, and proteins into plant cells and tissues. Moreover, we highlight the exploiting of CPPs as advantageous and beneficial tool for plant genome editing via delivery of nuclease proteins, and provide a practical example of genome alternation through CPP-delivered nucleases. Finally, the current exploitation of peptides in organelle-specific DNA delivery and modification of organellar genomes is discussed.

Secretome Analysis of Clavibacter nebraskensis Strains Treated with Natural Xylem Sap In Vitro Predicts Involvement of Glycosyl Hydrolases and Proteases in Bacterial Aggressiveness.

The Gram-positive bacterium Clavibacter nebraskensis (Cn) causes Goss's wilt and leaf blight on corn in the North American Central Plains with yield losses as high as 30%. Cn strains vary in aggressiveness on corn, with highly aggressive strains causing much more serious symptoms and damage to crops. Since Cn inhabits the host xylem, we investigated differences in the secreted proteomes of Cn strains to determine whether these could account for phenotypic differences in aggressiveness. Highly and a weakly aggressive Cn strains (Cn14-15-1 and DOAB232, respectively) were cultured, in vitro, in the xylem sap of corn (CXS; host) and tomato (TXS; non-host). The secretome of the Cn strains were extracted and processed, and a comparative bottom-up proteomics approach with liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to determine their identities and concentration. Relative quantitation of peptides was based on precursor ion intensities to measure protein abundances. In total, 745 proteins were identified in xylem sap media. In CXS, a total of 658 and 396 proteins were identified in strains Cn14-5-1 and DOAB232, respectively. The unique and the differentially abundant proteins in the secretome of strain Cn14-5-1 were higher in either sap medium compared to DOAB232. These proteins were sorted using BLAST2GO and assigned to 12 cellular functional processes. Virulence factors, e.g., cellulase, ß-glucosidase, ß-galactosidase, chitinase, ß-1,4-xylanase, and proteases were generally higher in abundance in the aggressive Cn isolate. This was corroborated by enzymatic activity assays of cellulase and protease in CXS. These proteins were either not detected or detected at significantly lower abundance levels in Cn strains grown in non-host xylem sap (tomato), suggesting potential factors involved in Cn-host (corn) interactions.

MinION Nanopore-based detection of Clavibacter nebraskensis, the corn Goss's wilt pathogen, and bacteriomic profiling of necrotic lesions of naturally-infected leaf samples.

The Goss's bacterial wilt pathogen, Clavibacter nebraskensis, of corn is a candidate A1 quarantine organism; and its recent re-emergence and spread in the USA and Canada is a potential biothreat to the crop. We developed and tested an amplicon-based Nanopore detection system for C. nebraskensis (Cn), targeting a purine permease gene. The sensitivity (1 pg) of this system in mock bacterial communities (MBCs) spiked with serially diluted DNA of C. nebraskensis NCPPB 2581T is comparable to that of real-time PCR. Average Nanopore reads increased exponentially from 125 (1pg) to about 6000 reads (1000 pg) after a 3-hr run-time, with 99.0% of the reads accurately assigned to C. nebraskensis. Three run-times were used to process control MBCs, Cn-spiked MBCs, diseased and healthy leaf samples. The mean Nanopore reads doubled as the run-time is increased from 3 to 6 hrs while from 6 to 12 hrs, a 20% increment was recorded in all treatments. Cn-spiked MBCs and diseased corn leaf samples averaged read counts of 5,100, 11,000 and 14,000 for the respective run-times, with 99.8% of the reads taxonomically identified as C. nebraskensis. The control MBCs and healthy leaf samples had 47 and 14 Nanopore reads, respectively. 16S rRNA bacteriomic profiles showed that Sphingomonas (22.7%) and Clavibacter (21.2%) were dominant in diseased samples while Pseudomonas had only 3.5% relative abundance. In non-symptomatic leaf samples, however, Pseudomonas (20.0%) was dominant with Clavibacter at 0.08% relative abundance. This discrepancy in Pseudomonas abundance in the samples was corroborated by qPCR using EvaGreen chemistry. Our work outlines a new useful tool for diagnosis of the Goss's bacterial wilt disease; and provides the first insight on Pseudomonas community dynamics in necrotic leaf lesions.

Verticillium dahliae's Isochorismatase Hydrolase Is a Virulence Factor That Contributes to Interference With Potato's Salicylate and Jasmonate Defense Signaling.

This study aimed to dissect the function of the Isochorismatase Hydrolase (ICSH1) gene in Verticillium dahliae's pathogenesis on potato. VdICSH1 was up-regulated in V. dahliae after induction with extracts from potato tissues. Its expression increased more in response to root extracts than to leaf and stem extracts. However, such expression in response to root extracts was not significantly different in the highly and weakly aggressive isolates tested. During infection of detached potato leaves, VdICSH1 expression increased significantly in the highly aggressive isolate compared to the weakly aggressive one. We generated icsh1 mutants from a highly aggressive isolate of V. dahliae and compared their pathogenicity with that of the original wild type strain. The analysis showed that this gene is required for full virulence of V. dahliae on potatoes. When we previously found differential accumulation of ICSH1 protein in favor of the highly aggressive isolate, as opposed to the weakly aggressive one, we had hypothesized that ICSH would interfere with the host's defense SA-based signaling. Here, we measured the accumulation of both salicylic acid (SA) and jasmonic acid (JA) in potato plants inoculated with an icsh1 mutant in comparison with the wild type strain. The higher accumulation of bound SA in the leaves in response to the icsh1 mutant compared to the wild type confirms the hypothesis that ICSH1 interferes with SA. However, the different trends in SA and JA accumulation in potato in the roots and in the stems at the early infection stages compared to the leaves at later stages indicate that they are both associated to potato defenses against V. dahliae. The expression of members of the isochorismatase family in the icsh1 mutants compensate that of ICSH1 transcripts, but this compensation disappears in presence of the potato leaf extracts. This study indicates ICSH1's involvement in V. dahliae's pathogenicity and provides more insight into its alteration of the SA/JA defense signaling's networking.

Molecular cloning, functional characterization and expression of potato (Solanum tuberosum) 1-deoxy-d-xylulose 5-phosphate synthase 1 (StDXS1) in response to Phytophthora infestans.

1-Deoxy-D-xylulose 5-phosphate synthase (DXS) catalyzes the initial step of the plastidial 2C-methyl-D-erythritol-4-phosphate (DOXP-MEP) pathway involved in isoprenoid biosynthesis. In this study, we cloned the complete cDNA of potato DXS gene that was designated StDXS1. StDXS1 cDNA encodes for 719 amino acid residues, with MW of 77.8 kDa, and is present in one copy in the potato genome. Phylogenetic analysis and protein sequence alignments assigned StDXS1 to a group with DXS homologues from closely related species and exhibited homodomain identity with known DXS proteins from other plant species. Late blight symptoms occurred in parallel with a reduction in StDXS1 transcript levels, which may be associated with the levels of isoprenoids that contribute to plant protection against pathogens. Subcellular localization indicated that StDXS1 targets the chloroplasts where isoprenoids are synthesized. Arabidopsis expressing StDXS1 showed a higher accumulation of carotenoids and chlorophyll as compared to wild type controls. Lower levels of ABA and GA were detected in the transgenic DXS lines as compared to control plants, which reflected on higher germination rates of the transgenic DXS lines. No changes were detected in JA or SA contents. Selected downstream genes in the DOXP-MEP pathway, especially GGPPS genes, were up-regulated in the transgenic lines.

Biochemical and molecular characterization of barley plastidial ADP-glucose transporter (HvBT1).

In cereals, ADP-glucose transporter protein plays an important role in starch biosynthesis. It acts as a main gate for the transport of ADP-glucose, the main precursor for starch biosynthesis during grain filling, from the cytosol into the amyloplasts of endospermic cells. In this study, we have shed some light on the molecular and biochemical characteristics of barley plastidial ADP-glucose transporter, HvBT1. Phylogenetic analysis of several BT1 homologues revealed that BT1 homologues are divided into two distinct groups. The HvBT1 is assigned to the group that represents BT homologues from monocotyledonous species. Some members of this group mainly work as nucleotide sugar transporters. Southern blot analysis showed the presence of a single copy of HvBT1 in barley genome. Gene expression analysis indicated that HvBT1 is mainly expressed in endospermic cells during grain filling; however, low level of its expression was detected in the autotrophic tissues, suggesting the possible role of HvBT1 in autotrophic tissues. The cellular and subcellular localization of HvBT1 provided additional evidence that HvBT1 targets the amyloplast membrane of the endospermic cells. Biochemical characterization of HvBT1 using E. coli system revealed that HvBT1 is able to transport ADP-glucose into E. coli cells with an affinity of 614.5 µM and in counter exchange of ADP with an affinity of 334.7 µM. The study also showed that AMP is another possible exchange substrate. The effect of non-labeled ADP-glucose and ADP on the uptake rate of [α-32P] ADP-glucose indicated the substrate specificity of HvBT1 for ADP-glucose and ADP.