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The nonstructural protein NSm of tomato spotted wilt virus (TSWV) has been identified as the avirulence determinant of the tomato single dominant Sw‐5 resistance gene. Although Sw‐5 effectiveness has been shown for most TSWV isolates, the emergence of resistance‐breaking (RB) isolates has been observed. It is strongly associated with two point mutations (C118Y or T120N) in the NSm viral protein. TSWV‐like symptoms were observed in tomato crop cultivars (+Sw‐5) in the Baja California peninsula, Mexico, and molecular methods confirmed the presence of TSWV. Sequence analysis of the NSm 118–120 motif and three‐dimensional protein modelling exhibited a noncanonical C118F substitution in seven isolates, suggesting that this substitution could emulate the C118Y‐related RB phenotype. Furthermore, phylogenetic and molecular analysis of the full‐length genome (TSWV‐MX) revealed its reassortment‐related evolution and confirmed that putative RB‐related features are restricted to the NSm protein. Biological and mutational NSm 118 residue assays in tomato (+Sw‐5) confirmed the RB nature of TSWV‐MX isolate, and the F118 residue plays a critical role in the RB phenotype. The discovery of a novel TSWV‐RB Mexican isolate with the presence of C118F substitution highlights a not previously described viral adaptation in the genus Orthotospovirus, and hence, the necessity of further crop monitoring to alert the establishment of novel RB isolates in cultivated tomatoes. We discovered a novel tomato spotted wilt virus (TSWV) isolate that encodes an NSm protein displaying a noncanonical C118F amino acid substitution, which is associated with Sw‐5 tomato gene resistance breaking.

P. aeruginosa is classified as a priority one pathogen by the World Health Organisation, and new drugs are urgently needed, due to the emergence of multidrug-resistant (MDR) strains. Antimicrobial-resistant nosocomial pathogens such as P. aeruginosa pose unwavering and increasing threats. Antimicrobial stewardship has been a challenge during the COVID-19 pandemic, with a majority of those hospitalized with SARS-CoV2 infection given antibiotics as a safeguard against secondary bacterial infection. This increased usage, along with increased handling of sanitizers and disinfectants globally, may further accelerate the development and spread of cross-resistance to antibiotics. In addition, P. aeruginosa is the primary causative agent of morbidity and mortality in people with the life-shortening genetic disease cystic fibrosis (CF). Prolonged periods of selective pressure, associated with extended antibiotic treatment and the actions of host immune effectors, results in widespread adaptive and acquired resistance in P. aeruginosa found colonizing the lungs of people with CF. This review discusses the arsenal of resistance mechanisms utilized by P. aeruginosa , how these operate under high-stress environments such as the CF lung and how their interconnectedness can result in resistance to multiple antibiotic classes. Intrinsic, adaptive and acquired resistance mechanisms will be described, with a focus on how each layer of resistance can serve as a building block, contributing to multi-tiered resistance to antimicrobial activity. Recent progress in the development of anti-resistance adjuvant therapies, targeting one or more of these building blocks, should lead to novel strategies for combatting multidrug resistant P. aeruginosa. Anti-resistance adjuvant therapy holds great promise, not least because resistance against such therapeutics is predicted to be rare. The non-bactericidal nature of anti-resistance adjuvants reduce the selective pressures that drive resistance. Anti-resistance adjuvant therapy may also be advantageous in facilitating efficacious use of traditional antimicrobials, through enhanced penetration of the antibiotic into the bacterial cell. Promising anti-resistance adjuvant therapeutics and targets will be described, and key remaining challenges highlighted. As antimicrobial stewardship becomes more challenging in an era of emerging and re-emerging infectious diseases and global conflict, innovation in antibiotic adjuvant therapy can play an important role in extending the shelf-life of our existing antimicrobial therapeutic agents.

Sw-5b is a widely used resistance gene in tomato breeding to control tomato spotted wilt virus (TSWV). The NSm protein encoded by TSWV is identified as the avirulence (AVR) determinant in Sw-5b-mediated resistance. In the last decades, Sw-5b resistance breaking (RB) isolates were found and identified in many locations around the world. The resistance-breaking phenotype in all the previously verified TSWV Sw-5b RB strains is associated with the NSmC118Y or NSmT120N mutations. In the summer of 2022, a Sw-5b RB TSWV strain was recognized in a greenhouse in Hungary. In inoculation experiments this strain was able to infect tomato plants with the Sw-5b resistance gene. Molecular analysis of the NSm avirulence determinant revealed a single alteration in the NSm protein, D122G mutation was identified. To our knowledge, this is the first report to identify this amino acid alteration associated with resistance-breaking phenotype in Sw-5b resistant tomato plants.

Summary The cap‐binding protein eIF4E, through its interaction with eIF4G, constitutes the core of the eIF4F complex, which plays a key role in the circularization of mRNAs and their subsequent cap‐dependent translation. In addition to its fundamental role in mRNA translation initiation, other functions have been described or suggested for eIF4E, including acting as a proviral factor and participating in sexual development. We used CRISPR/Cas9 genome editing to generate melon eif4e knockout mutant lines. Editing worked efficiently in melon, as we obtained transformed plants with a single‐nucleotide deletion in homozygosis in the first eIF4E exon already in a T0 generation. Edited and non‐transgenic plants of a segregating F2 generation were inoculated with Moroccan watermelon mosaic virus (MWMV); homozygous mutant plants showed virus resistance, while heterozygous and non‐mutant plants were infected, in agreement with our previous results with plants silenced in eIF4E. Interestingly, all homozygous edited plants of the T0 and F2 generations showed a male sterility phenotype, while crossing with wild‐type plants restored fertility, displaying a perfect correlation between the segregation of the male sterility phenotype and the segregation of the eif4e mutation. Morphological comparative analysis of melon male flowers along consecutive developmental stages showed postmeiotic abnormal development for both microsporocytes and tapetum, with clear differences in the timing of tapetum degradation in the mutant versus wild‐type. An RNA‐Seq analysis identified critical genes in pollen development that were down‐regulated in flowers of eif4e/eif4e plants, and suggested that eIF4E‐specific mRNA translation initiation is a limiting factor for male gametes formation in melon. Resistance to MWMV is associated with eIF4E editing and eif4e knock‐out plants show a post‐meiotic, sporophytic male sterility phenotype.

Tomato cultivation across the world is severely affected by emerging plant viruses. An effective method for protection of commercial crops against viral threats is the use of cultivars harboring resistance genes. Tomato brown rugose fruit virus (ToBRFV), a recently emerged tobamovirus, is able to overcome the dominant resistance that is present in the majority of commercial tomato cultivars. In an effort to alleviate the severe consequences of ToBRFV on tomato production, tomato breeding companies are developing new cultivars with varying levels of resistance against ToBRFV. In the present study, cultivars with a new resistant phenotype against ToBRFV were screened against a wild-type isolate of ToBRFV, and subsequently, their performance under commercial greenhouse conditions was monitored. Following the identification of ToBRFV symptoms in a commercial greenhouse-where both new resistant and susceptible cultivars were interplanted-these cultivars were more closely examined. The presence of ToBRFV was molecularly confirmed on both cultivar types suggesting that the new resistance had been broken. High-throughput sequencing (HTS) was used to study the complete genomes of viral isolates present in the two cultivar types. The analysis revealed a single amino acid change at position 82 of the movement protein of ToBRFV in the isolate present in the new resistant cultivar compared with the isolate identified in the susceptible cultivar. A screening bioassay, that was performed to compare the infectivity of the two ToBRFV isolates, confirmed that only the isolate with this specific amino acid change could successfully infect the resistant cultivar, overcoming the new resistance against ToBRFV.

Summary Plants use intracellular nucleotide‐binding leucine‐rich repeat immune receptors (NLRs) to recognize pathogen‐encoded effectors and initiate immune responses. Tomato spotted wilt virus (TSWV), which has been found to infect >1000 plant species, is among the most destructive plant viruses worldwide. The Sw‐5b is the most effective and widely used resistance gene in tomato breeding to control TSWV. However, broad application of tomato cultivars carrying Sw‐5b has resulted in an emergence of resistance‐breaking (RB) TSWV. Therefore, new effective genes are urgently needed to prevent further RB TSWV outbreaks. In this study, we conducted artificial evolution to select Sw‐5b mutants that could extend the resistance spectrum against TSWV RB isolates. Unlike regular NLRs, Sw‐5b detects viral elicitor NSm using both the N‐terminal Solanaceae‐specific domain (SD) and the C‐terminal LRR domain in a two‐step recognition process. Our attempts to select gain‐of‐function mutants by random mutagenesis involving either the SD or the LRR of Sw‐5b failed; therefore, we adopted a stepwise strategy, first introducing a NSmRB‐responsive mutation at the R927 residue in the LRR, followed by random mutagenesis involving the Sw‐5b SD domain. Using this strategy, we obtained Sw‐5bL33P/K319E/R927A and Sw‐5bL33P/K319E/R927Q mutants, which are effective against TSWV RB carrying the NSmC118Y or NSmT120N mutation, and against other American‐type tospoviruses. Thus, we were able to extend the resistance spectrum of Sw‐5b; the selected Sw‐5b mutants will provide new gene resources to control RB TSWV.

Plant resistance, which acts as a selective pressure that affects viral population fitness, leads to the emergence of resistance‐breaking virus strains. Most recessive resistance to potyviruses is related to the mutation of eukaryotic translation initiation factor 4E (eIF4E) or its isoforms that break their interactions with the viral genome‐linked protein (VPg). In this study, we found that the VPg α1–α2 loop, which is essential for binding eIF4E, is the most variable domain of papaya ringspot virus (PRSV) VPg. PRSV VPg with the naturally occurring amino acid substitution of K105Q or E108G in the α1–α2 loop fails to interact with watermelon (Citrullus lanatus) eIF4E but interacts with watermelon eIF(iso)4E instead. Moreover, PRSV carrying these mutations can break the eIF4E‐mediated resistance to PRSV in watermelon accession PI 244019. We further revealed that watermelon eIF(iso)4E with the amino acid substitutions of DNQS to GAAA in the cap‐binding pocket could not interact with PRSV VPg with natural amino acid substitution of K105Q or E108G. Therefore, our finding provides a precise target for engineering watermelon germplasm resistant to resistance‐breaking PRSV isolates. Papaya ringspot virus isolates with amino acid substitution of K105Q or E108G in VPg can break the eIF4ED71G‐mediated resistance by re‐hijacking another susceptibility factor eIF(iso)4E in watermelon.

Beet necrotic yellow vein virus (BNYVV) causes rhizomania disease in sugar beet ( Beta vulgaris ), which is controlled since more than two decades by cultivars harboring the Rz1 resistance gene. The development of resistance-breaking strains has been favored by a high selection pressure on the soil-borne virus population. Resistance-breaking is associated with mutations at amino acid positions 67-70 (tetrad) in the RNA3 encoded pathogenicity factor P25 and the presence of an additional RNA component (RNA5). However, natural BNYVV populations are highly diverse making investigations on the resistance-breaking mechanism rather difficult. Therefore, we applied a reverse genetic system for BNYVV (A type) to study Rz1 resistance-breaking by direct agroinoculation of sugar beet seedlings. The bioassay allowed a clear discrimination between susceptible and Rz1 resistant plants already four weeks after infection, and resistance-breaking was independent of the sugar beet Rz1 genotype. A comprehensive screen of natural tetrads for resistance-breaking revealed several new mutations allowing BNYVV to overcome Rz1 . The supplementation of an additional RNA5 encoding the pathogenicity factor P26 allowed virus accumulation in the Rz1 genotype independent of the P25 tetrad. This suggests the presence of two distinct resistance-breaking mechanisms allowing BNYVV to overcome Rz1 . Finally, we showed that the resistance-breaking effect of the tetrad and the RNA5 is specific to Rz1 and has no effect on the stability of the second resistance gene Rz2 . Consequently, double resistant cultivars ( Rz1 + Rz2 ) should provide effective control of Rz1 resistance-breaking strains. Our study highlights the flexibility of the viral genome allowing BNYVV to overcome host resistance, which underlines the need for a continuous search for alternative resistance genes.

Tomato spotted wilt (TSW) disease caused by tomato spotted wilt orthotospovirus (TSWV, Orthotospovirus tomatomaculae ) poses a significant threat to specialty and staple crops worldwide by causing over a billion dollars in crop losses annually. Current strategies for TSWV diagnosis heavily rely on nucleic acid or protein-based techniques which require significant technical expertise, and are invasive, time-consuming, and expensive, thereby catalyzing the search for better alternatives. In this study, we explored the potential of Raman spectroscopy (RS) in early detection of TSW in a non-invasive and non-destructive manner. Specifically, we investigated whether RS could be used to detect strain specific TSW symptoms associated with four TSWV strains infecting three differentially resistant tomato cultivars. In the acquired spectra, we observed notable reductions in the intensity of vibrational peaks associated with carotenoids. Using high-performance liquid chromatography (HPLC), we confirmed that TSWV caused a substantial decrease in the concentration of lutein that was detected by RS. Finally, we demonstrated that Partial Least Squares-Discriminant Analysis (PLS-DA) could be used to differentiate strain-specific TSW symptoms across all tested cultivars. These results demonstrate that RS can be a promising solution for early diagnosis of TSW, enabling timely disease intervention and thereby mitigating crop losses inflicted by TSWV.

Sw‐5b is an effective resistance gene used widely in tomato to control tomato spotted wilt virus (TSWV), which causes severe losses in crops worldwide. Sw‐5b confers resistance by recognizing a 21‐amino‐acid peptide region of the viral movement protein NSm (NSm21, amino acids 115–135). However, C118Y or T120N mutation within this peptide region of NSm has given rise to field resistance‐breaking (RB) TSWV isolates. To investigate the potential ability of TSWV to break Sw‐5b‐mediated resistance, we mutagenized each amino acid on NSm21 and determined which amino acid mutations would evade Sw‐5b recognition. Among all alanine‐scan mutants, NSmP119A, NSmW121A, NSmD122A, NSmR124A, and NSmQ126A failed to induce a hypersensitive response (HR) when coexpressed with Sw‐5b in Nicotiana benthamiana leaves. TSWV with the NSmP119A, NSmW121A, or NSmQ126A mutation was defective in viral cell‐to‐cell movement and systemic infection, while TSWV carrying the NSmD122A or NSmR124A mutation was not only able to infect wild‐type N. benthamiana plants systemically but also able to break Sw‐5b‐mediated resistance and establish systemic infection on Sw‐5b‐transgenic N. benthamiana plants. Two improved mutants, Sw‐5bL33P/K319E/R927A and Sw‐5bL33P/K319E/R927Q, which we recently engineered and which provide effective resistance against field RB isolates carrying NSmC118Y or NSmT120N mutations, recognized all NSm21 alanine‐substitution mutants and conferred effective resistance against new experimental RB TSWV with the NSmD122A or NSmR124A mutation. Collectively, we determined the key residues of NSm for Sw‐5b recognition, investigated their potential RB ability, and demonstrated that the improved Sw‐5b mutants could provide effective resistance to both field and potential RB TSWV isolates. Two artificially improved Sw‐5b resistance gene mutants confer effective resistance to potential resistance‐breaking isolates of tomato spotted wilt orthotospovirus.