Genome-informed trophic classification and functional characterization of virulence proteins from the maize tar spot pathogen Phyllachora maydis.

Phyllachora maydis is an ascomycete foliar fungal pathogen and the causal agent of tar spot in maize. Though P. maydis is considered an economically important foliar pathogens of maize, our general knowledge of the trophic lifestyle and functional role of effector proteins from this fungal pathogen remains limited. Here, we utilized a genome-informed approach to predict the trophic lifestyle of P. maydis and functionally characterized a subset of candidate effectors from this fungal pathogen. Leveraging the most recent P. maydis genome annotation and the CATAStrophy pipeline, we show this fungal pathogen encodes a predicted Carbohydrate-active enzymes (CAZymes) repertoire consistent with that of biotrophs. To investigate fungal pathogenicity, we selected 18 candidate effector proteins that were previously shown to be expressed during primary disease development. We assessed whether these putative effectors share predicted structural similarity with other characterized fungal effectors and determined whether any suppress plant immune responses. Using AlphaFold2 and Foldseek, we showed one candidate effector, PM02_g1115, adopts a predicted protein structure similar to that of an effector from Verticillium dahlia. Furthermore, transient expression of candidate effector-fluorescent protein fusions in Nicotiana benthamiana revealed two putative effectors, PM02_g378 and PM02_g2610, accumulated predominantly in the cytosol, and three candidate effectors, PM02_g1115, PM02_g7882, and PM02_g8240 consistently attenuated chitin-mediated reactive oxygen species production. Collectively, these results presented herein provide insights into the predicted trophic lifestyle and putative functions of effectors from P. maydis and will likely stimulate continued research to elucidate the molecular mechanisms used by P. maydis to induce tar spot.

Regulation of plant immunity and growth by tomato receptor-like cytoplasmic kinase TRK1.

The molecular mechanisms of quantitative resistance (QR) to fungal pathogens and their relationships with growth pathways are poorly understood. We identified tomato TRK1 (TPK1b Related Kinase1) and determined its functions in tomato QR and plant growth. TRK1 is a receptor-like cytoplasmic kinase that complexes with tomato LysM Receptor Kinase (SlLYK1). SlLYK1 and TRK1 are required for chitin-induced fungal resistance, accumulation of reactive oxygen species, and expression of immune response genes. Notably, TRK1 and SlLYK1 regulate SlMYC2, a major transcriptional regulator of jasmonic acid (JA) responses and fungal resistance, at transcriptional and post-transcriptional levels. Further, TRK1 is also required for maintenance of proper meristem growth, as revealed by the ectopic meristematic activity, enhanced branching, and altered floral structures in TRK1 RNAi plants. Consistently, TRK1 interacts with SlCLV1 and SlWUS, and TRK1 RNAi plants show increased expression of SlCLV3 and SlWUS in shoot apices. Interestingly, TRK1 suppresses chitin-induced gene expression in meristems but promotes expression of the same genes in leaves. SlCLV1 and TRK1 perform contrasting functions in defense but similar functions in plant growth. Overall, through molecular and biochemical interactions with critical regulators, TRK1 links upstream defense and growth signals to downstream factor in fungal resistance and growth homeostasis response regulators.

Candidate Effector Proteins from the Maize Tar Spot Pathogen Phyllachora maydis Localize to Diverse Plant Cell Compartments.

Most fungal pathogens secrete effector proteins into host cells to modulate their immune responses, thereby promoting pathogenesis and fungal growth. One such fungal pathogen is the ascomycete Phyllachora maydis, which causes tar spot disease on leaves of maize (Zea mays). Sequencing of the P. maydis genome revealed 462 putatively secreted proteins, of which 40 contain expected effector-like sequence characteristics. However, the subcellular compartments targeted by P. maydis effector candidate (PmEC) proteins remain unknown, and it will be important to prioritize them for further functional characterization. To test the hypothesis that PmECs target diverse subcellular compartments, cellular locations of super yellow fluorescent protein-tagged PmEC proteins were identified using a Nicotiana benthamiana-based heterologous expression system. Immunoblot analyses showed that most of the PmEC-fluorescent protein fusions accumulated protein in N. benthamiana, indicating that the candidate effectors could be expressed in dicot leaf cells. Laser-scanning confocal microscopy of N. benthamiana epidermal cells revealed that most of the P. maydis putative effectors localized to the nucleus and cytosol. One candidate effector, PmEC01597, localized to multiple subcellular compartments including the nucleus, nucleolus, and plasma membrane, whereas an additional putative effector, PmEC03792, preferentially labelled both the nucleus and nucleolus. Intriguingly, one candidate effector, PmEC04573, consistently localized to the stroma of chloroplasts as well as stroma-containing tubules (stromules). Collectively, these data suggest that effector candidate proteins from P. maydis target diverse cellular organelles and could thus provide valuable insights into their putative functions, as well as host processes potentially manipulated by this fungal pathogen.

Tomato PEPR1 ORTHOLOG RECEPTOR-LIKE KINASE1 Regulates Responses to Systemin, Necrotrophic Fungi, and Insect Herbivory.

Endogenous peptides regulate plant immunity and growth. Systemin, a peptide specific to the Solanaceae, is known for its functions in plant responses to insect herbivory and pathogen infections. Here, we describe the identification of the tomato (Solanum lycopersicum) PEPR1/2 ORTHOLOG RECEPTOR-LIKE KINASE1 (PORK1) as the TOMATO PROTEIN KINASE1b (TPK1b) interacting protein and demonstrate its biological functions in systemin signaling and tomato immune responses. Tomato PORK1 RNA interference (RNAi) plants with significantly reduced PORK1 expression showed increased susceptibility to tobacco hornworm (Manduca sexta), reduced seedling growth sensitivity to the systemin peptide, and compromised systemin-mediated resistance to Botrytis cinerea. Systemin-induced expression of Proteinase Inhibitor II (PI-II), a classical marker for systemin signaling, was abrogated in PORK1 RNAi plants. Similarly, in response to systemin and wounding, the expression of jasmonate pathway genes was attenuated in PORK1 RNAi plants. TPK1b, a key regulator of tomato defense against B. cinerea and M. sexta, was phosphorylated by PORK1. Interestingly, wounding- and systemin-induced phosphorylation of TPK1b was attenuated when PORK1 expression was suppressed. Our data suggest that resistance to B. cinerea and M. sexta is dependent on PORK1-mediated responses to systemin and subsequent phosphorylation of TPK1b. Altogether, PORK1 regulates tomato systemin, wounding, and immune responses.

Comparative Analysis of Host Range, Ability to Infect Tomato Cultivars with Tm-22 Gene, and Real-Time Reverse Transcription PCR Detection of Tomato Brown Rugose Fruit Virus.

Tomato (Solanum lycopersicum L.) is one of the most important vegetables in the world. However, tomato is also susceptible to many viral diseases. Several tobamoviruses, including tomato mosaic virus (ToMV), tomato mottle mosaic virus (ToMMV), and tomato brown rugose fruit virus (ToBRFV), are highly contagious pathogens that could result in significant economic losses if not controlled effectively. Tobamoviruses have been managed relatively well with broad adaptation of tomato cultivars with resistance genes. However, emergence of ToBRFV was shown to break down resistance conferred by the common resistance genes, resulting in serious outbreaks in many countries in Asia, Europe, and North America. The objective of this study was to conduct a comparative analysis of biological properties, including host range and disease resistance of ToMV, ToMMV, and ToBRFV. Results showed that despite many similarities in the host range, there were some unique host plant responses for each of the three viruses. In a comparative evaluation of disease resistance using the same tomato cultivars with or without Tm-22 gene, there was a striking difference in responses from tomato plants with Tm-22 gene inoculated with ToBRFV, ToMV, or ToMMV. Whereas these test plants were resistant to ToMV or ToMMV infection, all test plants were susceptible to ToBRFV. Further, for ToBRFV detection, a sensitive and reliable multiplex real-time reverse transcription (RT)-PCR assay using TaqMan probe with an internal 18S rRNA control was also developed. With simple modifications to RNA extraction and seed soaking, real-time RT-PCR could consistently detect the virus in single infested seed in varied levels of contamination, suggesting its usefulness for seed health assay.

Evaluation of Tomato Germplasm against Tomato Brown Rugose Fruit Virus and Identification of Resistance in Solanum pimpinellifolium.

The tomato is one of the most important vegetable crops grown worldwide. Tomato brown rugose fruit virus (ToBRFV), a seed-borne tobamovirus, poses a serious threat to tomato production due to its ability to break the resistant genes (Tm-1, Tm-2, Tm-22) in tomatoes. The objective of this work was to identify new resistant source(s) of tomato germplasm against ToBRFV. To achieve this aim, a total of 476 accessions from 12 Solanum species were tested with the ToBRFV US isolate for their resistance and susceptibility. As a result, a total of 44 asymptomatic accessions were identified as resistant/tolerant, including thirty-one accessions of S. pimpinellifolium, one accession of S. corneliomulleri, four accessions of S. habrochaites, three accessions of S. peruvianum, and five accessions of S. subsection lycopersicon hybrid. Further analyses using serological tests identified four highly resistant S. pimpinellifolium lines, PI 390713, PI 390714, PI 390716, and PI 390717. The inheritance of resistance in the selected lines was verified in the next generation and confirmed using RT-qPCR. To our knowledge, this is a first report of high resistance to ToBRFV in S. pimpinellifolium. These new genetic resources will expand the genetic pool available for breeders to develop new resistant cultivars of tomato against ToBRFV.

Comparative Analysis of Tomato Brown Rugose Fruit Virus Isolates Shows Limited Genetic Diversity.

Tomato is an important vegetable in the United States and around the world. Recently, tomato brown rugose fruit virus (ToBRFV), an emerging tobamovirus, has impacted tomato crops worldwide and can result in fruit loss. ToBRFV causes severe symptoms, such as mosaic, puckering, and necrotic lesions on leaves; other symptoms include brown rugose and marbling on fruits. More importantly, ToBRFV can overcome resistance in tomato cultivars carrying the Tm-22 locus. In this study, we recovered ToBRFV sequences from tomato seeds, leaves, and fruits from the U.S., Mexico, and Peru. Samples were pre-screened using a real-time RT-PCR assay prior to high-throughput sequencing. Virus draft genomes from 22 samples were assembled and analyzed against more than 120 publicly available genomes. Overall, most sequenced isolates were similar to each other and did not form a distinct population. Phylogenetic analysis revealed three clades within the ToBRFV population. Most of the isolates (95%) clustered in clade 3. Genetic analysis revealed differentiation between the three clades indicating minor divergence occurring. Overall, pairwise identity showed limited genetic diversity among the isolates in this study with worldwide isolates, with a pairwise identity ranging from 99.36% and 99.97%. The overall population is undergoing high gene flow and population expansion with strong negative selection pressure at all ToBRFV genes. Based on the results of this study, it is likely that the limited ToBRFV diversity is associated with the rapid movement and eradication of ToBRFV-infected material between countries.

The Botrytis cinerea Crh1 transglycosylase is a cytoplasmic effector triggering plant cell death and defense response.

Crh proteins catalyze crosslinking of chitin and glucan polymers in fungal cell walls. Here, we show that the BcCrh1 protein from the phytopathogenic fungus Botrytis cinerea acts as a cytoplasmic effector and elicitor of plant defense. BcCrh1 is localized in vacuoles and the endoplasmic reticulum during saprophytic growth. However, upon plant infection, the protein accumulates in infection cushions; it is then secreted to the apoplast and translocated into plant cells, where it induces cell death and defense responses. Two regions of 53 and 35 amino acids are sufficient for protein uptake and cell death induction, respectively. BcCrh1 mutant variants that are unable to dimerize lack transglycosylation activity, but are still able to induce plant cell death. Furthermore, Arabidopsis lines expressing the bccrh1 gene exhibit reduced sensitivity to B. cinerea, suggesting a potential use of the BcCrh1 protein in plant immunization against this necrotrophic pathogen.

Phytochemicals and antioxidative enzymes defence mechanism on occurrence of yellow vein mosaic disease of pumpkin (Cucurbita moschata).

Pumpkin (Cucurbita moschata) samples showing yellow vein mosaic disease in Varanasi region were identified with begomovirus infection using PCR amplification. A sequencing analysis of the full genome revealed that it is a strain of Tomato leaf curl Palampur virus (GenBank ID. FJ931537). Phytochemical composition and antioxidative enzyme levels were compared in infected and healthy plants. The study revealed that the amount of total protein declined in the infected leaves but elevated up to 135 % in the fruits of infected plants, whereas vitamin C and antioxidants declined in infected leaves as well as fruits. There was substantial increase in total phenol content in leaves (72 %) and fruits (300 %) of infected plants. In infected samples, substantial increase in activities of superoxide dismutases (SOD), ascorbate peroxidase (APX), guaiacol peroxidase (GPX) and catalase (CAT) was observed as compared to the uninfected control plants. The native PAGE showed alterations in the intensities of isozyme bands in the infected plants. The APX, GPX, CAT, SOD and glutamate dehydrogenase (GDH) bands were intense in the infected plants, whereas the GR isozyme showed reduced intensity in diseased plants.