Susceptibility Of Mutants Research Articles

Screening 60Co-γ Irradiated Camellia oleifera Lines for Anthracnose-Resistant

Camellia oleifera C. Abel is a woody oil crop with multiple purposes. This study aims to investigate the mutagenic effects of 60Co-γ radiation on C. oleifera seedlings and to screen anthracnose-resistant mutants. Two C. oleifera varieties were investigated: ‘Xianglin 1’ (XL1) and ‘Xianglin 210’ (XL210). Seeds were irradiated with 0 Gy, 30 Gy, 50 Gy, and 80 Gy of 60Co-γ, and after one year of planting, the mutagenic lines were studied, and disease-resistant mutants were screened. Results showed that as the radiation intensity was increased, the emergence percentage of both C. oleifera XL210 and XL1 was significantly decreased. Radiation significantly changed the SOD and POD activities in both varieties. Furthermore, 80 Gy irradiated lines showed reduced anthracnose resistance in both varieties. However, 50 Gy irradiated lines showed enhanced disease resistance in XL210 while reducing it in XL1. The 30 Gy irradiated lines did not affect the disease resistance of either variety. Colletotrichum gloeosporioides infection tests were conducted on 94 mutant C. oleifera seedlings, resulting in 8 highly resistant mutants (A3, A8, A10, A19, A21, A32, A35, B17) and 3 susceptible mutants (A4, B15, B27) in XL210 and XL1. Differences in SOD and POD activities led to variations in disease resistance among different mutants. Additionally, the expression levels of CoSOD1, CoPOD, CoIDD4, and CoWKRY78 were varied among the different mutants. This study delivers the screening of disease-resistant mutants in C. oleifera through mutagenic breeding, providing material for the development of new C. oleifera varieties and serving as a resource for further research in mutagenic breeding.

Comparative Transcriptomics of Soybean Genotypes with Partial Resistance Toward Phytophthora sojae, Conrad, and M92-220 to Moderately Susceptible Fast Neutron Mutant Soybeans and Sloan.

The breeding of disease-resistant soybeans cultivars to manage Phytophthora root and stem rot caused by the pathogen Phytophthora sojae involves combining quantitative disease resistance (QDR) and Rps gene-mediated resistance. To identify and confirm potential mechanisms of QDR toward P. sojae, we conducted a time course study comparing changes in gene expression among Conrad and M92-220 with high QDR to susceptible genotypes, Sloan, and three mutants derived from fast neutron irradiation of M92-220. Differentially expressed genes from Conrad and M92-220 indicated several shared defense-related pathways at the transcriptomic level but also defense pathways unique to each cultivar, such as stilbenoid, diarylheptanoid, and gingerol biosynthesis and monobactam biosynthesis. Gene Ontology pathway analysis showed that the susceptible fast neutron mutants lacked enrichment of three terpenoid-related pathways and two cell wall-related pathways at either one or both time points, in contrast to M92-220. The susceptible mutants also lacked enrichment of potentially important Kyoto Encyclopedia of Genes and Genomes pathways at either one or both time points, including sesquiterpenoid and triterpenoid biosynthesis; thiamine metabolism; arachidonic acid; stilbenoid, diarylheptanoid, and gingerol biosynthesis; and monobactam biosynthesis. Additionally, 31 genes that were differentially expressed in M92-220 following P. sojae infection were not expressed in the mutants. These 31 genes have annotations related to unknown proteins; valine, leucine, and isoleucine biosynthesis; and protein and lipid metabolic processes. The results of this study confirm previously proposed mechanisms of QDR, provide evidence for potential novel QDR pathways in M92-220, and further our understanding of the complex network associated with QDR mechanisms in soybean toward P. sojae.

Rice Brown Planthopper, Nilaparvata lugens (Stål) Feeding Behaviour in Resistant NAGINA22 Rice Mutants

Background: Rice (Oryza sativa L.), is an important staple food and an excellent source of calories in India. Of the different insect pests attacking rice, brown planthopper, BPH, Nilaparvata lugens (Stål) is an important sucking pest causing upto 100% yield losses. Host plant resistance is the most important method in keeping BPH under control and understanding the underlying mechanisms of resistance is necessary to breed the resistant varieties with desirable characters. Methods: Selected EMS mutant lines of Nagina22 (N22) variety, which were previously mass screened under controlled greenhouse conditions were assessed for their antixenosis mechanism to brown planthopper feeding by measuring probing marks and honeydew excretion area by following standard procedures and statistical analysis was done by using Statistix 8.1 software. Result: Both the brown planthopper nymphs and adults probed more in the resistant mutants compared to the susceptible mutants whereas the BPH adults probed more times (24.4) compared to the nymphs (22.2). The recorded honeydew excretion area by the resistant mutants was lower than that of susceptible mutants. In general, the BPH adults fed more and excreted more honeydew (79.5 mm2) compared to the nymphs (59.0 mm2). The damage score has a negative correlation with the probing marks of adults (-0.207) and nymphs (-0.411); and a positive correlation with honeydew excretion of adults (0.547) and nymphs (0.200). In this study, the EMS N22 mutants resistant to BPH, with a greater number of probing marks and less honeydew excretion area can serve as best donors in the breeding programmes to develop brown planthopper resistant varieties.

Mcl-1 mediates intrinsic resistance to RAF inhibitors in mutant BRAF papillary thyroid carcinoma

Papillary thyroid carcinoma (PTC) is the most frequent form of thyroid cancer. PTC commonly presents with mutations of the serine/threonine kinase BRAF (BRAFV600E), which drive ERK1/2 pathway activation to support growth and suppress apoptosis. PTC patients often undergo surgical resection; however, since the average age of PTC patients is under 50, adverse effects associated with prolonged maintenance therapy following total thyroidectomy are a concern. The development of mutant-selective BRAF inhibitors (BRAFi), like vemurafenib, has been efficacious in patients with metastatic melanoma, but the response rate is low for mutant BRAF PTC patients. Here, we assay the therapeutic response of BRAFi in a panel of human PTC cell lines and freshly biopsied patient samples. We observed heterogeneous responses to BRAFi, and multi-omic comparisons between susceptible and resistant mutant BRAF PTC revealed overrepresented stress response pathways and the absence of compensatory RTK activation – features that may underpin innate resistance. Importantly, resistant cell lines and patient samples had increased hallmarks of failed apoptosis; a cellular state defined by sublethal caspase activation and DNA damage. Further analysis suggests that the failed apoptotic phenotypes may have features of “minority mitochondrial outer membrane permeabilization (MOMP)” – a stress-related response characterized by fragmented and porous mitochondria known to contribute to cancer aggressiveness. We found that cells presenting with minority MOMP-like phenotypes are dependent on the apoptotic regulator, Mcl-1, as treatment with the Mcl-1 inhibitor, AZD5991, potently induced cell death in resistant cells. Furthermore, PI3K/AKT inhibitors sensitized resistant cells to BRAFi; an effect that was at least in part associated with reduced Mcl-1 levels. Together, these data implicate minority MOMP as a mechanism associated with intrinsic drug resistance and underscore the benefits of targeting Mcl-1 in mutant BRAF PTC.

Exploring mechanisms of drought-tolerance and adaptation of selected sesame mutant lines

Sesame (Sesamum indicum L.) is an important oilseed crop, but its productivity and quality are often compromised by drought stress. Developing drought-resistant sesame varieties is thus crucial for its sustainable cultivation. In this study, we conducted a comprehensive investigation of morphoanatomical and physiochemical mechanisms underlying drought tolerance in selected sesame mutants. The drought-tolerant mutants ‘ML2-37’ and ‘ML2-72’ were compared to the susceptible mutant ‘US1-2’ and the parental cultivar ‘ML13’ under controlled greenhouse conditions. Our results showed that the tolerant mutants exhibited superior performance in response to drought stress. We identified specific adaptation mechanisms, including accumulation of proline, glycine, and cuticular wax, which contribute to maintaining optimal water status. Additionally, the tolerant mutants showed a significant reduction in the number and diameter of the xylem vessels, even under severe stress. These results open up good perspectives for the development of drought-resistant sesame varieties, thus offering a promising solution to improve agricultural productivity in regions affected by increasing water scarcity in the actual context of climate change. The selection indices proposed in this study provide practical tools to identify and develop sesame germplasm and cultivars adapted to drought stress conditions. This work constitutes a significant contribution to research on drought tolerance in sesame and offers promising prospects for sustainable agriculture in the context of global climate change.

Fruits from tomato carotenoid mutants have altered susceptibility to grey mold

The necrotrophic fungus Botritys cinerea takes advantage of the oxidative burst to facilitate tissue infection, leading to substantial losses during tomato postharvest. Tomato fruit is a source of carotenoids, pigments with a wide variety of isomeric configurations that determine their antioxidant capacity. Here, fruit susceptibility to B. cinerea was assessed in Micro-Tom Near Isogenic lines harboring mutations that alter the profile of carotenoids. Wound-inoculated fruit of the mutants Delta carotene (Del) and tangerine (t), which show large variety of carotenoids rather than the major accumulation of trans-lycopene, were less susceptible to the pathogen. Differences in susceptibility between the mutants were only observed in ripe fruit, after the formation of carotenoids, and they were associated with attenuation of damage caused by reactive oxygen species. The greater variety of carotenoid isomers, which in turn contributed to the greater lipophilic antioxidant capacity of fruit, was associated with the less susceptible mutants, Del and t. Together, our data reveals a potential activity of carotenoids in fruit defense, in addition to the well-known and widespread ecological role as attractors of seed dispersers.

Dissection of a rapidly evolving wheat resistance gene cluster by long-read genome sequencing accelerated the cloning of Pm69

Gene cloning in repeat-rich polyploid genomes remains challenging. Here, we describe a strategy for overcoming major bottlenecks in cloning of the powdery mildew resistance gene (R-gene) Pm69 derived from tetraploid wild emmer wheat. A conventional positional cloning approach was not effective owing to suppressed recombination. Chromosome sorting was compromised by insufficient purity. A Pm69 physical map, constructed by assembling Oxford Nanopore Technology (ONT) long-read genome sequences, revealed a rapidly evolving nucleotide-binding leucine-rich repeat (NLR) R-gene cluster with structural variations. A single candidate NLR was identified by anchoring RNA sequencing reads from susceptible mutants to ONT contigs and was validated by virus-induced gene silencing. Pm69 is likely a newly evolved NLR and was discovered in only one location across the wild emmer wheat distribution range in Israel. Pm69 was successfully introgressed into cultivated wheat, and a diagnostic molecular marker was used to accelerate its deployment and pyramiding with other R-genes.

Mutation and sequencing-based cloning and functional studies of a rust resistance gene in sunflower (Helianthus annuus).

Rust, caused by the fungus Puccinia helianthi Schwein., is one of the most devastating diseases of sunflower (Helianthus annuus L.), affecting global production. The rust R gene R11 in sunflower line HA-R9 shows broad-spectrum resistance to P. helianthi virulent races and was previously mapped to an interval on sunflower chromosome 13 encompassing three candidate genes annotated in the XRQr1.0 reference genome assembly. In the current study, we combined ethyl methane sulfonate (EMS) mutagenesis with targeted region capture and PacBio long-read sequencing to clone the R11 gene. Sequencing of a 60-kb region spanning the R11 locus from the R11 -HA-R9 rust-resistant line and three EMS-induced susceptible mutants facilitated the identification of R11 and definition of induced mutations. The R11 gene is predicted to have a single 3996-bp open reading frame and encodes a protein of 1331 amino acids with CC-NBS-LRR domains typical of genes conferring plant resistance to biotrophic pathogens. Point mutations identified in the R11 rust-susceptible mutants resulted in premature stop codons, consistent with loss of function leading to rust susceptibility. Additional functional studies using comparative RNA sequencing of the resistant line R11 -HA-R9 and R11 -susceptible mutants revealed substantial differences in gene expression patterns associated with R11 -mediated resistance at 7 days post-inoculation with rust, and uncovered the potential roles of terpenoid biosynthesis and metabolism in sunflower rust resistance.

The reverse mutation of CsMLO8 results in susceptibility to powdery mildew via inhibiting cell wall apposition formation and cell death in cucumber (Cucumis sativus L.)

Powdery mildew (PM) is an obligate biotrophic pathogen that requires living cells to grow and reproduce, causing huge losses in cucumbers. However, the resistance mechanism against PM in cucumber remains poorly understood. In this study, a susceptible mutant m6326 was found from the EMS-mutagenized population of No.26. Genetic analysis of the F2 population and sequence analysis showed that No.26 was a natural mlo mutant, and m6326 was a CsMLO8 revertant. The phytopathological investigation and transcriptome analysis of the resistant line (No.26) and susceptible revertant (m6326) after PM infection were carried out. The higher rate of spore penetration, longer hypha and more conidia in m6326 were observed than that in No.26. Histological analysis showed that the formation of cell wall apposition and cell death in No.26 leaves was faster and more than that in m6326 after powdery mildew infection. Transcriptome analysis identified differentially expressed genes between the resistant and susceptible lines. The phenylpropanoid biosynthesis pathway and ROS scavenging genes were significantly up-regulated in No.26 compared with that in m6326. Our findings suggested that the reverse mutation of CsMLO8 results in susceptibility to powdery mildew, which was related to cell wall apposition formation and cell death.

CRISPR/Cas9 based mlo-mediated resistance against Podosphaera xanthii in cucumber (Cucumis sativus L.).

Powdery mildews (PM) are common and severe pathogen groups that threaten plants, and PM resistance is complex and polygenic in cucumbers. Previously mlo-based resistance was reported in various plants, including cucumber, with generated loss-of CsaMLO function mutants. However, mlo-based resistance in cucumber is also complex and involves additional mechanisms such as hypersensitive response (HR) and papillae formation. For this reason, we focused on determining the mlo-based powdery mildew resistance mechanism in cucumber. CRISPR/Cas9 was used in the present study to generate loss-of-function mutants for CsaMLO1, CsaMLO8, and CsaMLO11 of PM susceptible ADR27 cucumber inbred lines and CsaMLO mutants were obtained and validated. Trypan Blue and DAB staining were performed to detect Podosphaera xanthii germination/penetration rates and accumulation of Reactive Oxygen Species (ROS). Our results indicate that PM-susceptibility associated CsaMLOs in cucumber are negative regulators in different defense mechanisms against powdery mildew at early and late stages of infection. Further, the experiment results indicated that CsaMLO8 mutation-based resistance was associated with the pre-invasive response, while CsaMLO1 and CsaMLO11 could be negative regulators in the post-invasive defense response in cucumber against P. xanthii. Although the loss-of CsaMLO8 function confers the highest penetration resistance, CsaMLO1 and CsaMLO11 double mutations could be potential candidates for HR-based resistance against PM pathogen in cucumber. These results highlighted the crucial role of CRISPR/Cas9 to develop PM resistant cucumber cultivars, possessing strong pre-invasive defense with CsaMLO8 or post-invasive with CsaMLO1/CsaMLO11 mutations.

Fine Mapping of Stripe-Rust-Resistance Gene YrJ22 in Common Wheat by BSR-Seq and MutMap-Based Sequencing

Identification and accurate mapping of new resistance genes are essential for gene pyramiding in wheat breeding. The YrJ22 gene is a dominant stripe-rust-resistance gene located at the distal end of chromosome 2AL, which was identified in a leading Chinese-wheat variety, Jimai 22, showing high resistance to CYR32, a prevalent race of Puccinia striiformis tritici (Pst) in China. In the current study, 15 F1 and 2273 F2 plants derived from the cross of Jimai 22/Avocet S were used for the fine-mapping of YrJ22. The RNA-Seq of resistant and susceptible bulks of F2 plants (designated BSR-Seq) identified 10 single-nucleotide polymorphisms (SNP) in a 12.09 Mb physical interval on chromosome 2AL. A total of 1022 EMS-induced M3 lines of Jimai 22 were screened, to identify susceptible mutants for MutMap analysis. Four CAPS markers were developed from SNPs identified using BSR-Seq and MutMap. A linkage map for YrJ22 was constructed with 11 CAPS/STS and three SSR markers. YrJ22 was located at a 0.9 cM genetic interval flanked by markers H736 and H400, corresponding to a 340.46 kb physical region (768.7-769.0 Mb), including 13 high-confidence genes based on the Chinese Spring reference genome. TraesCS2A01G573200 is a potential candidate-gene, according to linkage and quantitative real-time PCR (qPCR) analyses. The CAPS marker H732 designed from an SNP in TraesCS2A01G573200 co-segregated with YrJ22. These results provide a useful stripe-rust-resistance gene and molecular markers for marker-assisted selection in wheat breeding and for further cloning of the gene.

Orthologous genes Pm12 and Pm21 from two wild relatives of wheat show evolutionary conservation but divergent powdery mildew resistance

Wheat powdery mildew, caused by Blumeria graminis f. sp. tritici (Bgt), is a devastating disease that threatens wheat production worldwide. Pm12, which originated from Aegilops speltoides, a wild relative of wheat, confers strong resistance to powdery mildew and therefore has potential use in wheat breeding. Using susceptible mutants induced by gamma irradiation, we physically mapped and isolated Pm12 and showed it to be orthologous to Pm21 from Dasypyrum villosum, also a wild relative of wheat. The resistance function of Pm12 was validated via ethyl methanesulfonate mutagenesis, virus-induced gene silencing, and stable genetic transformation. Evolutionary analysis indicates that the Pm12/Pm21 loci in wheat species are relatively conserved but dynamic. Here, we demonstrated that the two orthologous genes, Pm12 and Pm21, possess differential resistance against the same set of Bgt isolates. Overexpression of the coiled-coil domains of both PM12 and PM21 induces cell death in Nicotiana benthamiana leaves. However, their full-length forms display different cell death-inducing activities caused by their distinct intramolecular interactions. Cloning of Pm12 will facilitate its application in wheat breeding programs. This study also gives new insight into two orthologous resistance genes, Pm12 and Pm21, which show different race specificities and intramolecular interaction patterns.

Different adaptive patterns of wheat with different drought tolerance under drought stresses and rehydration revealed by integrated metabolomic and transcriptomic analysis.

Wheat as a staple food crop is enduring ever-frequent intermittent and changing drought with the climate change. It is of great significance to highlight the adaptive approaches under such variable conditions at multiple levels to provide a comprehensive understanding of drought tolerance and facilitate the genetic breeding of wheat. Therefore, three wheat lines with different drought tolerance (drought-tolerant mutant Mu > common wheat CK > drought susceptible mutant mu) were analyzed under moderate and severe drought stresses as well as rehydration. Samples were subjected to transcriptomic and metabolomic profiling in combination with physiological and biochemical determination. The moderate drought stress rendered 198 and 115 differentially expressed metabolites (DEMs) in CK and Mu, respectively. The severe drought stress rendered 166, 151 and 137 DEMs in CK, Mu and mu, respectively. The rehydration rendered 150 and 127 DEMs in CK and Mu. 12,557 and 10,402 differentially expressed genes (DEGs) were identified for CK and Mu under moderate drought stress, respectively. 9,893, 7,924, and 9,387 DEGs were identified for CK, Mu, and mu under severe drought stress, respectively. 13,874 and 14,839 were identified in CK and Mu under rehydration, respectively. Metabolomics results showed that amino acid was the most differentially expressed metabolites, followed by phenolic acids. Flavonoids played an important role in drought tolerance. Most enriched pathways under drought included biosynthesis of secondary metabolites, metabolic pathways and photosynthesis. Metabolites and genes involved in osmotic regulation, antioxidase activities, and ABA signaling were more enriched in Mu than in CK and mu. Various drought-responsive genes and metabolites in Mu showed different trends with those in CK and mu. Increased amino acids biosynthetic capability and ROS scavenging ability resulted from higher antioxidase activities and increased flavonoids may be the mechanisms underlying the drought tolerance characteristic of Mu. Recovery from reversible ROS damage and rapid amino acid biosynthesis may contribute to the rapid recovery of Mu. The present study provides new insights for mechanisms of wheat under complex drought conditions.

Large-scale mutational analysis of wheat powdery mildew resistance gene Pm21.

Wheat powdery mildew is a devastating disease leading to severe yield loss. The powdery mildew resistance gene Pm21, encoding a nucleotide-binding leucine-rich repeat receptor (NLR) protein, confers broad-spectrum resistance to powdery mildew and has great potential for controlling this disease. In this study, a large-scale mutagenesis was conducted on wheat cultivar (cv.) Yangmai 18 carrying Pm21. As a result, a total of 113 independent mutant lines susceptible to powdery mildew were obtained, among which, only one lost the whole Pm21 locus and the other 112 harbored one- (107) or two-base (5) mutations in the encoding region of Pm21. From the 107 susceptible mutants containing one-base change, we found that 25 resulted in premature stop codons leading to truncated proteins and 82 led to amino acid changes involving in 59 functional sites. We determined the mutations per one hundred amino acids (MPHA) indexes of different domains, motifs, and non-domain and non-motif regions of PM21 protein and found that the loss-of-function mutations occurred in a tendentious means. We also observed a new mutation hotspot that was closely linked to RNBS-D motif of the NB-ARC domain and relatively conserved in different NLRs of wheat crops. In addition, we crossed all the susceptible mutants with Yangmai 18 carrying wild-type Pm21, subsequently phenotyped their F1 plants and revealed that the variant E44K in the coiled-coil (CC) domain could lead to dominant-negative effect. This study revealed key functional sites of PM21 and their distribution characteristics, which would contribute to understanding the relationship of resistance and structure of Pm21-encoded NLR.

Field evaluation of promising mutants of mungbean against Fela Beetle, Aphid and POD Borer

Experiment was conducted at the Agricultural Farm of Patuakhali Science and Technology University to know the morphological characteristics of different mutants of mungbean and to find out tolerant/least susceptible mutant(s) against flea beetle, aphid and pod borer based on the reaction of insect pests to promising mutants of mungbean during the period from April to June, 2015. The mutant MBM-07(S)-2 was found to be the tallest (32.57 cm) with highest number of branches/plant (5.03), highest number of leaves/plant (11.06) among all mutants and check variety while MBM-07-Y-2 (23.40 cm) was the shortest plant height, MBM-656-51-2 had the lowest number of branches/plant (3.73), MBM-347-13 (7.03) had the lowest number of leaves/plant. The mutant MBM-427-87-3 had the highest number of pods (5.00) compared to other mutants but MBM-656-51-2 had the lowest number of pods/plant (3.09). Significantly the highest pod length and the highest number of seeds/pod were recorded in the mutant MBM-07-Y-1 (6.37 cm) while the lowest pod length (5.19 cm) was in MBM-80(LCAL) and lowest number of seeds/pod was in the mutant MBM-390-94-Y (5.34). Mutant MBM-347-13 had the lowest population of flea beetle (18.00) and pod borer (2.00) compared to all other mutants while the lowest number of aphid/plant was observed in mutant MBM-07-Y-1 (1.00). In case of infestation, the mutant MBM-347-13 had the lowest percentage of infestation (20.69%) by flea beetle, (3.18%) by pod borer but the lowest infestation by aphid (3.33%) was recorded in the mutant MBM-390-94-Y while the mutant MBM-427-87-3 had the highest percentage of plant infestation (37.80%) by flea beetle, pod damage (9.69%) by pod borer and variety BARI moog-6 had the highest percentage (34.45%) of plant infestation by aphid. Mutant MBM-347-13 was least susceptible to flea beetle and pod borer while mutant MBM-427-87-3 was highly susceptible to flea beetle and pod borer. However, mutant MBM-390-94-Yand MBM-07-Y-2 were less susceptible to aphid while BARI moog-6 and mutant MBM-427-87-3 were highly susceptible to aphid. SAARC J. Agric., 20(1): 97-106 (2022)

Genetic detection and identification of the candidate resistance gene for a major QTL resistant to Puccinia polysora in maize

Southern corn rust (SCR) is a major foliar disease in maize. Identification the major QTL and candidate resistance gene is prerequisite in maize resistance breeding programs. To identify major resistance loci/gene(s) resistant to SCR, a recombinant inbred line (RIL) population derived from a cross between 975 and 12 (resistant) and Lx9801 (susceptible) was analyzed. In total four QTLs were identified, which were located on chromosomes 5, 9 and 10, respectively. Among them, a major QTL on chromosome 10 (qSCR3), were detected in all the environments, which can explain 70.3–78.4% of the total phenotypic variation. Fine-mapping of qSCR3 delimited the QTL to an interval of ∼225 Kb. Using transcripts assembly of the 975–12 and a susceptible mutant (975-12S), an NLR gene, Zm00025ab420970, was identified as the candidate gene for qSCR3, which is a new allele/gene resistant to SCR in maize. Since qSCR3 is a major QTL resistant to SCR, it can be used as a desirable resistance source in maize resistance breeding programs.

Bet-hedging antimicrobial strategies in macrophage phagosome acidification drive the dynamics of Cryptococcus neoformans intracellular escape mechanisms.

The fungus Cryptococcus neoformans is a major human pathogen with a remarkable intracellular survival strategy that includes exiting macrophages through non-lytic exocytosis (Vomocytosis) and transferring between macrophages (Dragotcytosis) by a mechanism that involves sequential events of non-lytic exocytosis and phagocytosis. Vomocytosis and Dragotcytosis are fungal driven processes, but their triggers are not understood. We hypothesized that the dynamics of Dragotcytosis could inherit the stochasticity of phagolysosome acidification and that Dragotcytosis was triggered by fungal cell stress. Consistent with this view, fungal cells involved in Dragotcytosis reside in phagolysosomes characterized by low pH and/or high oxidative stress. Using fluorescent microscopy, qPCR, live cell video microscopy, and fungal growth assays we found that the that mitigating pH or oxidative stress reduced Dragotcytosis frequency, whereas ROS susceptible mutants of C. neoformans underwent Dragotcytosis more frequently. Dragotcytosis initiation was linked to phagolysosomal pH, oxidative stresses, and macrophage polarization state. Dragotcytosis manifested stochastic dynamics thus paralleling the dynamics of phagosomal acidification, which correlated with the inhospitality of phagolysosomes in differently polarized macrophages. Hence, randomness in phagosomal acidification randomly created a population of inhospitable phagosomes where fungal cell stress triggered stochastic C. neoformans non-lytic exocytosis dynamics to escape a non-permissive intracellular macrophage environment.

Proline confers acid stress tolerance to Bacillus megaterium G18

Proline plays a multifunctional role in several organisms including bacteria in conferring protection under stress conditions. In this paper we report the role of proline in conferring acid tolerance to Bacillus megaterium G18. An acid susceptible mutant of B. megaterium G18 which required proline for its growth under acid stress condition was generated through Tn5 mutagenesis. Further, targeted inactivation of proC involved in osmo-adaptive proline synthesis in B. megaterium G18 resulted in the loss of ability of the bacterium to grow at low pH (pH 4.5). Exogenous supply of proline (1 mM) to the growth medium restored the ability of the mutant cells to grow at pH 4.5 which was not the same in case of other osmoprotectants tested. Proline was produced and secreted to extracellular medium by B. megaterium G18 when growing in low pH condition as evidenced by the use of Escherichia coli proline auxotrophs and HPLC analysis. Further, pHT01 vector based expression of full length proC gene in the ∆proC mutant cells restored the survival capacity of the mutant cells in acidic pH, suggesting that proline production is an important strategy employed by B. megaterium G18 to survive under acid stress induced osmotic stress.

Low-Level Cefepime Exposure Induces High-Level Resistance in Environmental Bacteria: Molecular Mechanism and Evolutionary Dynamics.

Antibiotics exert selective pressures on clinically relevant antibiotic resistance. It is critical to understand how antibiotic resistance evolves in environmental microbes exposed to subinhibitory concentrations of antibiotics and whether evolutionary dynamics and emergence of resistance are predictable. In this study, Comamonas testosteroni isolated from wastewater activated sludge were subcultured in a medium containing 10 ng/mL cefepime for 40 days (∼300 generations). Stepwise mutations were accumulated, leading to an ultimate 200-fold increase in the minimum inhibitory concentration (MIC) of cefepime. Early stage mutation in DNA polymerase-encoding gene dnaE2 played an important role in antibiotic resistance evolution. Diverse resistance mechanisms were employed and validated experimentally, including increased efflux, biofilm formation, reduced antibiotic uptake, and drug inactivation. The cefepime minimal selective concentrations (MSCs) and relative fitness of susceptible, intermediate, and resistant mutants were determined. Agent-based modeling of the modified Moran process enabled simulations of resistance evolution and predictions of the emergence time and frequency of resistant mutants. The unraveled cefepime resistance mechanisms could be employed by broader bacteria, and the newly developed model is applicable to the predictions of general resistance evolution. The improved knowledge facilitates the assessment, prediction, and mitigation of antibiotic resistance progression in antibiotic-polluted environments.

Development and Metabolic Characterization of Horse Gram (Macrotyloma uniflorum Lam. (Verdc.)) Mutants for Powdery Mildew Resistance

Horse gram is one of the lesser-known beans widely grown in India. One hundred and twenty-three homozygous horse gram mutants were screened for powdery mildew (PM) disease resistance using the grade 0 to 4. The mutants were grouped based on the disease level of 0 to 2 (resistant) and susceptible (3 to 4). The PM altered the chlorophyll fluorescence (a/b ratio), maturity duration, and yield attributing traits. The yield loss ranged from 4.55% to 72.66%. After affirming the resistance level, the resistant mutant (RM) with minimum yield loss (scale:0) and the susceptible mutant (SM) with maximum loss (scale:4) were used for metabolomic analysis through GC-MS. PM infection induced expression of 66 metabolites representing 32 functional classes. The number of unique classes in RM and SM was 13 and 11, respectively, while eight were common. A fold change in the common metabolites indicated an enhanced accumulation of amine, alcohol, and ester in RM. Along with pathogen-induced defensive metabolites, RM produced silane and fluorene, whose biological significance in disease resistance is unknown. Though SM expressed defence-related bio-molecules, it failed to yield better.