Optimizing RNAi-Target by Nicotiana benthamiana-Soybean Mosaic Virus System Drives Broad Resistance to Soybean Mosaic Virus in Soybean

Soybean mosaic virus (SMV) is a prevalent pathogen of soybean (Glycine max). Pyramiding multiple SMV-resistance genes into one individual is tedious and difficult, and even if successful, the obtained multiple resistance might be broken by pathogen mutation, while targeting viral genome via host-induced gene silencing (HIGS) has potential to explore broad-spectrum resistance (BSR) to SMV. We identified five conserved target fragments (CTFs) from S1 to S5 using multiple sequence alignment of 30 SMV genome sequences and assembled the corresponding target-inverted-repeat constructs (TIRs) from S1-TIR to S5-TIR. Since the inefficiency of soybean genetic transformation hinders the function verification of batch TIRs in SMV-resistance, the Nicotiana benthamiana-chimeric-SMV and N. benthamiana-pSMV-GUS pathosystems combined with Agrobacterium-mediated transient expression assays were invented and used to test the efficacy of these TIRs. From that, S1-TIR assembled from 462 bp CTF-S1 with 92% conservation rate performed its best on inhibiting SMV multiplication. Accordingly, S1-TIR was transformed into SMV-susceptible soybean NN1138-2, the resistant-healthy transgenic T1-plants were then picked out via detached-leaf inoculation assay with the stock-plants continued for progeny reproduction (T1 dual-utilization). All the four T3 transgenic progenies showed immunity to all the inoculated 11 SMV strains under individual or mixed inoculation, achieving a strong BSR. Thus, optimizing target for HIGS via transient N. benthamiana-chimeric-SMV and N. benthamiana-pSMV-GUS assays is crucial to drive robust resistance to SMV in soybean and the transgenic S1-TIR-lines will be a potential breeding source for SMV control in field.

Targeted and Untargeted Metabolomics Profiling of Wheat Reveals Amino Acids Increase Resistance to Fusarium Head Blight

Fusarium head blight (FHB), a notorious plant disease caused by Fusarium graminearum (F. graminearum), is severely harmful to wheat production, resulting in a decline in grain quality and yield. In order to develop novel control strategies, metabolomics has been increasingly used to characterize more comprehensive profiles of the mechanisms of underlying plant-pathogen interactions. In this research, untargeted and targeted metabolomics were used to analyze the metabolite differences between two wheat varieties, the resistant genotype Sumai 3 and the susceptible genotype Shannong 20, after F. graminearum inoculation. The untargeted metabolomics results showed that differential amino acid metabolic pathways existed in Sumai 3 and Shannong 20 after F. graminearum infection. Additionally, some of the amino acid contents changed greatly in different cultivars when infected with F. graminearum. Exogenous application of amino acids and F. graminearum inoculation assay showed that proline (Pro) and alanine (Ala) increased wheat resistance to FHB, while cysteine (Cys) aggravated the susceptibility. This study provides an initial insight into the metabolite differences of two wheat cultivars under the stress of F. graminearum. Moreover, the method of optimization metabolite extraction presents an effective and feasible strategy to explore the understanding of the mechanisms involved in the FHB resistance.

A Ubiquitously Expressed UDP-Glucosyltransferase, UGT74J1, Controls Basal Salicylic Acid Levels in Rice

Salicylic acid (SA) is a phytohormone that regulates a variety of physiological and developmental processes, including disease resistance. SA is a key signaling component in the immune response of many plant species. However, the mechanism underlying SA-mediated immunity is obscure in rice (Oryza sativa). Prior analysis revealed a correlation between basal SA level and blast resistance in a range of rice varieties. This suggested that resistance might be improved by increasing basal SA level. Here, we identified a novel UDP-glucosyltransferase gene, UGT74J1, which is expressed ubiquitously throughout plant development. Mutants of UGT74J1 generated by genome editing accumulated high levels of SA under non-stressed conditions, indicating that UGT74J1 is a key enzyme for SA homeostasis in rice. Microarray analysis revealed that the ugt74j1 mutants constitutively overexpressed a set of pathogenesis-related (PR) genes. An inoculation assay demonstrated that these mutants had increased resistance against rice blast, but they also exhibited stunted growth phenotypes. To our knowledge, this is the first report of a rice mutant displaying SA overaccumulation.

A ubiquitously expressed UDP-glucosyltransferase, UGT74J1, controls basal salicylic acid levels in rice

Salicylic acid (SA) is a phytohormone that regulates a variety of physiological and developmental processes, including disease resistance. SA is a key signaling component in the immune response of many plant species. However, the mechanism underlying SA-mediated immunity is obscure in rice (Oryza sativa). Prior analysis revealed a correlation between basal SA level and blast resistance in a range of rice varieties. This suggested that resistance might be improved by increasing basal SA level. Here, we identified a novel UDP-glucosyltransferase gene, UGT74J1, which is expressed ubiquitously throughout plant development. Mutants of UGT74J1 generated by genome editing accumulated high levels of SA under non-stressed conditions, indicating that UGT74J1 is a key enzyme for SA homeostasis in rice. Microarray analysis revealed that the ugt74j1 mutants constitutively overexpressed a set of pathogenesis-related (PR) genes. An inoculation assay demonstrated that these mutants had increased resistance against rice blast, but they also exhibited stunted growth phenotypes. To our knowledge, this is the first report of a rice mutant displaying SA overaccumulation.

The identification of Tautoneura mori as the vector of mulberry crinkle leaf virus and the infectivity of infectious clones in mulberry

Mulberry crinkle leaf virus (MCLV) is a novel geminivirus identified from mulberry. The pathogenicity and the natural vector of transmission have remained unknown for MCLV. Here, the infectious clones which consisted of the complete tandem dimeric genome of MCLV in a binary vector were constructed and agro-inoculated into mulberry seedlings. The results showed that the infectious clones of MCLV were systemically infectious to mulberry, but the infected mulberry plants did not show any virus-like symptoms. The natural transmission vectors of MCLV were also identified from possible vector insects occurring on the MCLV-infected mulberry plants. The vector ability of Tautoneura mori Matsumura was identified through inoculation assay. Three of 21 (14.3%) seedlings inoculated with T. mori collected from MCLV-infected mulberry plants grown naturally were detected to be MCLV-positive 50 days post-inoculation. These MCLV-positive mulberry plants did also not show any virus-like symptoms. Collectively, it is suggested that MCLV is infectious to mulberry plants, but MCLV alone does not induce symptoms. The leafhopper T. mori was for the first time determined experimentally to be a transmission vector of MCLV.

Extreme resistance to potato virus A in potato cultivar Barbara is independently mediated by Ra and Rysto

Potato virus A (PVA) and potato virus Y (PVY) are two common members of Potyvirus genus infecting potato crops worldwide. Host resistance offers an economical and effective means for the control and/or management of these viruses. In this study, 20 potato clones were screened for their resistance against PVA and PVY by mechanical and/or graft inoculation assay, and were explored for the relationship between extreme resistance genes Ra and Ry by the detection of molecular markers linked respectively to Ryadg, Rysto, and Rychc. Six clones, including Barbara, Jizhangshu 8, Longshu 7, Longshu 8, M6, and Solara, were found to be extremely resistant to both PVA and PVY; three clones (AC142, Eshu 3, and Shepody) were deemed to be extremely resistant to PVA but susceptible to PVY. To further reveal the inheritance of the extreme resistance (ER) against PVA, a tetraploid F1 population of Barbara × F58050 (susceptible to both PVY and PVA) and a tetraploid BC1 population of BF145 (a PVA-resistant but PVY-susceptible progeny of Barbara × F58050) × F58050 were obtained, and phenotyping of the F1 and BC1 population by graft-inoculation with PVA showed segregation ratios of 3:1 and 1:1 (R:S), respectively. These results suggested that two independent loci control ER against PVA in Barbara: one confers ER to both PVA and PVY, and the other confers ER to PVA only. The deduced genotype of Barbara is RyryryryRararara.

Validation of chip grafting inoculation assay to assess the resistance of Solanum species against phytoplasma

Big bud caused by several different phytoplasmas is an emerging threat to tomato production worldwide. The development of resistant varieties would be an effective approach to manage this problem, but it requires an appropriate screening technique. Recently, we have described a simple and efficient chip graft inoculation assay (CGIA) for the first time to screen tomato germplasm against Tomato leaf curl New Delhi virus. The present study was conducted to first validate the CGIA for phytoplasma transmission, then to assess the resistance of 74 genotypes belonging to different Solanum species against 16SrII-D phytoplasma. CGIA success rate and phytoplasma transmission was 100% since all the grafts survived and phytoplasma was detected in these plants using nested polymerase chain reaction. No genotype was found resistant as all the grafted plants showed typical disease symptoms. In addition to phytoplasma transmission, CGIA can be used for better understanding the plant–phytoplasma interactions and biology of phytoplasmas in tomato.

Evidence of apple blotch resistance in wild apple germplasm (Malus spp.) accessions

Diplocarpon coronariae causes apple blotch, which results in significant economic losses. Resistance to apple blotch in commercial apple cultivars has not been reported in recent literature. The Malus germplasm collection from the Fruit Genebank of the Julius Kühn-Institut that is maintained in Dresden, Germany consists of 516 accessions of about 49 Malus species. This apple gene pool was evaluated for resistance to Diplocarpon coronariae. A four-year field survey was subsequently followed by artificial inoculation of field-selected accessions. Fifteen accessions that represent nine different species and hybrids were confirmed to be highly resistant following a repeated inoculation assay in the laboratory. The results from this investigation is of high interest to breeders for future apple breeding programmes and investigations of resistance to Diplocarpon coronariae.