First Detection and Characterization of Cross- and Multiple Resistance to Acetyl-CoA Carboxylase (ACCase)- and Acetolactate Synthase (ALS)-Inhibiting Herbicides in Black-Grass (Alopecurus myosuroides) and Italian Ryegrass (Lolium multiflorum) Populations from Ireland

Understanding the resistance spectrum and underlying genetic mechanisms is critical for managing herbicide-resistant populations. In this study, resistance to acetyl CoA carboxylase (ACCase) and acetolactate synthase (ALS) inhibitors was investigated in four suspected resistant populations of Alopecurus myosuroides (ALOMY-001 to ALOMY-004) and Lolium multiflorum (LOLMU-001 to LOLMU-004), collected from cereal production fields in Ireland. Glasshouse assays with three ALOMY-active herbicides [propaquizafop, cycloxydim (ACCase) and mesosulfuron + iodosulfuron (ALS)] or five LOLMU-active herbicides [pinoxaden, propaquizafop, cycloxydim (ACCase) and mesosulfuron + iodosulfuron, pyroxsulam (ALS)], and target-site resistance mechanism studies, based on pyrosequencing, were carried out in each of those populations. For A. myosuroides, Ile-1781-Leu ACCase mutation contributed to propaquizafop and cycloxydim resistance (shoot dry weight GR50 resistance factor (RF) = 7.5–35.5) in all ALOMY populations, and the independent Pro-197-Thr or Pro-197-Ser ALS mutation contributed to mesosulfuron + iodosulfuron resistance (RF = 3.6–6.6), in ALOMY-002 to ALOMY-004. Most of the analyzed plants for these mutations were homo/heterozygous combinations or only heterozygous. For L. multiflorum, phenotypic resistance to mesosulfuron + iodosulfuron (RF = 11.9–14.6) and pyroxsulam (RF = 2.3–3.1) was noted in all LOLMU populations, but the Pro-197-Gln or Pro-197-Leu ALS mutation (mostly in homozygous status) was identified in LOLMU-001, LOLMU-002 and LOLMU-004 only. Additionally, despite no known ACCase mutations in any LOLMU populations, LOLMU-002 survived pinoxaden and propaquizafop application (RF = 3.4 or 1.3), and LOLMU-003 survived pinoxaden (RF = 2.3), suggesting the possibility of non-target-site resistance mechanisms for ACCase and/or ALS resistance in these populations. Different resistance levels, as evidenced by a reduction in growth as dose increased above field rates in ALOMY and LOLMU, were due to variations in mutation rate and the level of heterozygosity, resulting in an overall resistance rating of low to moderate. This is the first study confirming cross- and multiple resistance to ACCase- and ALS-inhibiting herbicides, highlighting that resistance monitoring in A. myosuroides and L. multiflorum in Ireland is critical, and the adoption of integrated weed management strategies (chemical and non-chemical/cultural strategies) is essential.

Single-Cell Sequencing Demonstrates Complex Resistance Landscape in CLL and MCL Treated with BTK and BCL2 Inhibitors

The genomic landscape of resistance to targeted agents (TAs) used as monotherapy in chronic lymphocytic leukemia (CLL) is complex and often heterogeneous at the patient level. To gain insight into the clonal architecture of acquired genomic resistance to BTK inhibitors and BCL2 inhibitors in CLL, particularly in patients carrying multiple resistance mutations, we performed targeted single-cell DNA sequencing of eight patients who developed progressive disease (PD) on TAs (either class). In all cases, analysis of single-cell architecture revealed mutual exclusivity between multiple resistance mutations to the same TA class, variable clonal co-occurrence of multiple mutations affecting different TAs in patients exposed to both classes, and a phenomenon of multiple independent emergences of identical nucleotide changes leading to canonical resistance mutations. We also report the first observation of established BCL2 resistance mutations in a patient with mantle cell lymphoma (MCL) following PD on sequential monotherapy, implicating BCL2 as a venetoclax resistance mechanism in MCL. Taken together, these data reveal the significant clonal complexity of CLL and MCL progression on TAs at the nucleotide level and confirm the presence of multiple, clonally independent, mechanisms of TA resistance within each individual disease context.

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.

Co-conjugation of Virulence Plasmid and KPC Plasmid in a Clinical Klebsiella pneumoniae Strain

Carbapenem-resistant hypervirulent Klebsiella pneumoniae (CR-HvKP) strains have been increasingly reported, and it is important to understand the evolutionary mechanisms of these highly pathogenic and resistant bacterial pathogens. In this study, we characterized a ST11 carbapenem-resistant K. pneumoniae strain which harbored an IncFIB/IncHI1B type virulence plasmid and an IncFII/IncR type blaKPC–2-bearing plasmid. The virulence plasmid was found to be conjugative and harbored a 35-kbp fragment including aerobactin encoding cluster from virulence plasmid pLVPK and multiple resistance genes, resulting in a mosaic multi-drug resistance and virulence plasmid. This virulence plasmid could be transferred via conjugation to Escherichia coli and K. pneumoniae strains alone as well as together with the blaKPC–2-bearing plasmid. Co-transmission of virulence and blaKPC–2-bearing plasmids would directly convert a classic K. pneumoniae strain into CR-HvKP strain, leading to a sharp increase in the prevalence of CR-HvKP in clinical settings, which poses a great threat to human health.

Sumatran Fleabane (Erigeron sumatrensis) Resistant to PSI-Inhibitor Herbicides and Physiological Responses to Paraquat

Herbicide-resistant weed management is one of the greatest agricultural challenges in crop production. Thus, the quick identification of resistant-herbicide weeds is extremely important for management. This study aimed to evaluate resistance to PSI-inhibitor herbicides (diquat) of Sumatran Fleabane [(Erigeron sumatrensis (Retz.) E.Walker)] and physiological response to paraquat application. The research was conducted with two E. sumatrensis biotypes, one susceptible and the other with multiple resistance to herbicides from five different modes of action (glyphosate, paraquat, diuron, saflufenacil, and 2,4-D). A dose-response assay was carried out to evaluate herbicide resistance to diquat in paraquat-resistant E. sumatrensis biotype. The enzymatic activities of superoxide dismutase (SOD), catalase (CAT), and ascorbate peroxidase (APX), hydrogen peroxide (H2O2) content, and chlorophyll a fluorescence were measured in both biotypes after paraquat (400 g ai ha−1) application. The dose-response assay confirmed resistance of E. sumatrensis to diquat with resistance factor levels of 26-fold and 6-fold for LD50 and GR50 values, respectively, compared with the susceptible biotype. The accumulation of H2O2 occurred faster in the paraquat-susceptible biotype than in the resistant ones. Paraquat treatment caused an increase in SOD and APX activity in the susceptible biotype, but antioxidant enzyme activities were unaffected by paraquat in the resistant one at 5 hours after application (HAA). Chlorophyll a fluorescence increased along the first 4 HAA in both resistant and susceptible biotypes. However, at 24 HAA the resistant biotype showed a decline in fluorescence close to untreated plants while susceptible one died, which can be used to diagnose paraquat resistance at 24 HAA. There is confirmed resistance to diquat in a paraquat-resistant E. sumatrensis biotype. The paraquat-resistant biotype does not induce antioxidative enzymes, as a possible mechanism of resistance to paraquat, but shows a fast recovery of photosynthesis and continuous growth when subjected to paraquat, while the paraquat-susceptible biotype does not survive.

Fungi Resistance to Multissite Fungicides

Multisite fungicides have been used for many years in fruit and vegetable crops worldwide. Cases of the fungi resistance development to these fungicides have been rare. From the 2002 season onwards, with the outbreak of Asian soybean rust in Brazil, caused by Phakopsora pachyrhizi, site-specific fungicides became the main weapon for its control. From 2002 to 2011, penetrant mobile site-specific fungicides were used and until today in double (DMI + QoI) or triple (DMI + QoI + SDHI) co-formulatoons in an area of more than 30 million hectares and with three sprays per area. This resulted, as expected, in the fungus sensitivity reduction, today with cross and multiple resistance to those site-specific fungicides. From the 2011 season in an attempt to recover control that for some chemicals and mixtures reached < 30%, research was started with site-specific + multi-site mixtures, taking as example Phytophthora infestans resistance development to metalaxyl in Europe showinig long-lasting solution found by the addition of multisite mancozeb. It is expected that the effective life of site-specific + multi-site mixtures may be as long in controlling soybean rust as it has been for potato, tomato and grape downy mildews. This review presents the concepts involved in the sensitivity reduction to fungicides. Some fungal species and fungicides involved are listed. Considering the P. pachyrhizi sporulation potential, the great soybean area sprayed and the number of sprays per area mainly with site-specific co-formulations and the reduced area sprayed with multisites, we discuss the need for annual monitoring of P. pachyrhizi sensitivity to the these chemicals.

pH Homeostasis and Sodium Ion Pumping by Multiple Resistance and pH Antiporters in Pyrococcus furiosus

Multiple Resistance and pH (Mrp) antiporters are seven-subunit complexes that couple transport of ions across the membrane in response to a proton motive force (PMF) and have various physiological roles, including sodium ion sensing and pH homeostasis. The hyperthermophilic archaeon Pyrococcus furiosus contains three copies of Mrp encoding genes in its genome. Two are found as integral components of two respiratory complexes, membrane bound hydrogenase (MBH) and the membrane bound sulfane sulfur reductase (MBS) that couple redox activity to sodium translocation, while the third copy is a stand-alone Mrp. Sequence alignments show that this Mrp does not contain an energy-input (PMF) module but contains all other predicted functional Mrp domains. The P. furiosus Mrp deletion strain exhibits no significant changes in optimal pH or sodium ion concentration for growth but is more sensitive to medium acidification during growth. Cell suspension hydrogen gas production assays using the deletion strain show that this Mrp uses sodium as the coupling ion. Mrp likely maintains cytoplasmic pH by exchanging protons inside the cell for extracellular sodium ions. Deletion of the MBH sodium-translocating module demonstrates that hydrogen gas production is uncoupled from ion pumping and provides insights into the evolution of this Mrp-containing respiratory complex.