Natural Products and Antiviral Resistance

Viral diseases are minacious with the potential for causing pandemics and treatment is complicated because of their inherent ability to mutate and become resistant to drugs. Antiviral drug resistance is a persistent problem that needs continuous attention by scientists, medical professionals, and government agencies. To solve the problem, an in-depth understanding of the intricate interplay between causes of antiviral drug resistance and potential new drugs specifically natural products is imperative in the interest and safety of public health. This review delves into natural product as reservoir for antiviral agents with the peculiar potentials for addressing the complexities associated with multi-drug resistant and emerging viral strains. An evaluation of the mechanisms underlying antiviral drug activity, antiviral drug resistance is addressed, with emphasis on production of broad-spectrum antiviral agents from natural sources. There is a need for continued natural product-based research, identification of new species and novel compounds.

Identification and Evaluation of Insect and Disease Resistance in Transgenic Cry1Ab13-1 and NPR1 Maize

PCR detection,quantitative real-time PCR(q-RTPCR),outdoor insect resistance,and disease resistance identification were carried out for the detection of genetic stability and disease resistance through generations(T2,T3,and T4)in transgenic maize germplasms(S3002 and 349)containing the bivalent genes(insect resistance gene Cry1Ab13-1 and disease resistance gene NPR1)and their corresponding wild type.Results indicated that the target genes Cry1Ab13-1 and NPR1 were successfully transferred into both germplasms through tested generations;q-PCR confirmed the expression of Cry1Ab13-1 and NPR1 genes in roots,stems,and leaves of tested maize plants.In addition,S3002 and 349 bivalent gene-transformed lines exhibited resistance to large leaf spots and corn borer in the field evaluation compared to the wild type.Our study confirmed that Cry1Ab13-1 and NPR1 bivalent genes enhanced the resistance against maize borer and large leaf spot disease and can stably inherit.These findings could be exploited for improving other cultivated maize varieties.

Conventional and Molecular Approaches towards Genetic Improvement in Pigeonpea for Insects Resistance

Pigeonpea [Cajanus cajan (L.) Millspaugh] is an important food legume of the semi-arid tropics (SAT) sustaining livelihood of millions of people. Stagnant and unstable yield per hectare all over the world is the characteristic feature of this crop. This is primarily ascribed to its susceptibility/sensitivity to a number of biotic and abiotic factors. Among biotic factors, insects such as pod borer (Helicoverpa armigera), pod fly (Melanoagromyza obtusa) and spotted borer (Maruca vitrata) substantially damage the crop and result in significant economic losses. Management of these insects by genetic means has always been considered environment friendly approach. However, genetic improvement has always been impeded by limited genetic variability in the primary gene pool of pigeonpea. Wild species present in the secondary and tertiary gene pools have been reported to carry resistance for such insects. However, transfer of resistance through conventional backcrossing has not been much successful. It calls for gene introgression through marker assisted backcrossing (MABC) or advanced backcross breeding (AB breeding). In this review, we have attempted to assess the progress made through conventional and molecular breeding and suggested the ways to move further towards genetic enhancement for insects resistance in

Resistance Analysis of the Binary Insect-resistant Transgenic Soybean to Heliothis viriplaca

[Objective] The aim of the research was to analyze the resistance of binary insect-resistant transgenic soybean to Heliothis viriplaca.[Method]In this experiment, resistance analysis of the stabilized binary insect-resistant transgenic soybean to Heliothis viriplaca was conducted in lab and in field conditions.[Result] The results indicated that the leaves of insect-resistant transgenic soybeans T5-150 and T5-195 showed lighter damage than those of non-transgenic soybeans. Meanwhile, the Heliothis viriplaca larvae fed on leaves of these two transgenic soybeans were characterized by less leaf consumption, shortening survival day, slower development and less pupation.[Conclusion]It was concluded that insect-resistance of transgenic soybean to Heliothis viriplaca was increased dramatically and the research provided a reference for selecting binary insect-resistant transgenic soybean to Heliothis viriplaca.

Insect Resistance of Different Tissues of Transgenic Cotton to Spodoptera exigua(Hbner)

[Objective] The aim was to learn the resistance of different tissues and organs of transgenic cotton to Spodoptera exigua （Hbner）. [Method] Flowers,the 1st,the 3rd,the 6th and the 14th leaves from the top of 33B,GK12 and SGK321 were used to feed S. exigua neonates respectively. Survival larvae and dead ones were counted on the 3rd,the 7th,the 10th,the 16th and the 19th day; meanwhile,the pupae amount was recorded,and the pupae weight was measured at the 24th h after pupation. [Result] The survival curves,pupation rates and pupae weights of S. exigua feeding on different tissues of transgenic cotton were not significantly different from those of S. exigua feeding on the corresponding tissues of conventional cotton; pupation rate of S. exigua feeding on different leaves of the same cotton variety were not significantly different from each other,but all higher than that of S. exigua feeding on the flowers of that cotton; and there were no differences among pupation weights of S. exigua feeding on different leaves or flowers of the same cotton variety. [Conclusion] Transgenic cotton showed weak resistance to S. exigua. Hence,in the transgenic cotton fields,more attention should be paid to occurrence trend of S. exigua and its control.

Test of Insect-Resistance of Transgenic Poplar with CpTI Gene

Both non-transgenic hybrid triploid poplars [ (Populus tomentosa×P.bolleana)×P.tomentosa ] and transgenic ones expressing cowpea trypsin inhibitor were cut at the base of the stem to produce auxoblasts, and used as source of leaves for insect feeding trials performed on 3 major insect species of poplar, the forest tent caterpillar ( Malacosoma disstria L.), gypsy moth ( Lymantria dispar L.) and willow moth ( Stilpnotia candida Staudinger). The height and basal diameter of trees were measured by the end of that year (2000). The results indicated that the growth elements of transgenic poplars were not interfered by the incorporation of the CpTI gene. Intriguingly, the height and basal diameter of the clone TG04 were much greater than that of the control. The transgenic foliage consumed by insects induced the increase of larval mortality, and decrease of larval wet weight gain, faecal output, pupal weight and egg deposition. Among them 3 transgenic clones, TG04, TG07 and TG71 received special attention for their outstanding insect resistance compared with other transgenic clones, which showed that the CpTI gene in them was expressed more actively and stably than in others.

Developing transgenic maize(Zea mays L.) with insect resistance and glyphosate tolerance by fusion gene transformation

Using linker peptide LP4/2A for multiple gene transformation is considered to be an effective method to stack or pyramid several traits in plants. Bacillus thuringiensis（Bt） cry gene and epsps（5-enolpyruvylshikimate-3-phosphate synthase） gene are two important genes for culturing pest-resistant and glyphosate-tolerant crops. We used linker peptide LP4/2A to connect the Bt cry1 Ah gene with the 2m G2-epsps gene and combined the wide-used man A gene as a selective marker to construct one coordinated expression vector called p2 EPUHLAGN. The expression vector was transferred into maize by Agrobacterium tumefaciens-mediated transformation, and 60 plants were obtained, 40% of which were positive transformants. Molecular detection demonstrated that the two genes in the fusion vector were expressed simultaneously and spliced correctly in translation processing; meanwhile bioassay detection proved the transgenic maize had preferable pest resistance and glyphosate tolerance. Therefore, linker peptide LP4/2A provided a simple and reliable strategy for producing gene stacking in maize and the result showed that the fusion gene transformation system of LP4/2A was feasible in monocot plants.

CopE and TLR6 RNAi-mediated tomato resistance to western flower thrips

The western flower thrips(WFT;Frankliniella occidentalis)is a mesophyll cell feeder that damages many crops.Management of WFT is complex due to factors such as high fecundity,short reproduction time,ability to feed on a broad range of host plants,and broad pesticide resistance.These challenges have driven research into developing alternative pest control approaches for WFT.This study analyzed the feasibility of a biological control-based strategy to manage WFT using RNA interference(RNAi)-mediated silencing of WFT endogenous genes.For the delivery of RNAi,we developed transgenic tomato lines expressing double-stranded RNA(dsRNA)of coatomer protein subunit epsilon(CopE)and Toll-like receptor 6(TLR6)from WFT.These genes are involved in critical biological processes of WFT,and their dsRNA can be lethal to these insects when ingested orally.Adult WFT that fed on the transgenic dsRNAexpressing tomato flower stalk showed increased mortality compared with insects that fed on wild-type samples.In addition,WFT that fed on TLR6 and CopE transgenic tomato RNAi lines showed reduced levels of endogenous CopE and TLR6 transcripts,suggesting that their mortality was likely due to RNAi-mediated silencing of these genes.Thus,our findings demonstrate that transgenic tomato plants expressing dsRNA of TLR6 and CopE can be lethal to F.occidentalis,suggesting that these genes may be deployed to control insecticide-resistant WFT.

Screening of Broad-spectrum Resistance Resources against Rice Bacterial Leaf Streak

[ Objectives] Rice materials with broad-spectrum resistance against several pathogenic strains of Xanthomans oryzae pv. Oryzicola （Xooc） at multiple growth stages were screened, in order to provide reliable resistance sources for variety breeding against rice bacterial leaf streak. [ Methods] A total of 1 100 rice lines with rich genetic background were offered as selective objects, and highly susceptible cultivar Jingang 30 was used as the control. Inoculation was carried out with five strong pathogenic strains of Xooc through acupuncture method at various growth stages for resistance identification. [ Results] Fourteen disease-resistant materials were obtained through preliminary screening, accounting for 1.27% of the total materials. Nine materials moderately resistant to bacterial leaf streak were obtained via secondary screening, accounting for 0.82% of the total materials. Besides, three of them （ RL6, RL9 and RLL4） were resistant to a number of pathogenic strains of Xooc at three growth stages. In particular, RL16 presented broad-spectrum resistance to multi-strains with high resistance levels. Six of them （ RI2, RIA, RIS, RL8, RL11 and RL12） were resistant to single strain at single growth stage. [ Conclusions] The obtained three materials have broad-spectrum resistance to bacterial leaf streak at multiple growth stages, which can be served as an important source to cultivate disease-resistant rice varieties. RL6 maintains high broad-spectrum resistance at all growth stages, which can be used as a prior resistance source for rice variety breeding. Six materials obtained with resistance against bacterial leaf streak at particular growth stages can be used as candidate resistance sources.

Evaluation of cotton germplasm for morphological and biochemical host plant UPdates resistance traits against sucking insect pests complex

Background:Sucking insect pests cause severe damage to cotton crop production.The development of insect resistant cotton cultivars is one of the most effective measures in curtailing the yield losses.Considering the role of morphological and biochemical host plant resista nee(HPR)traits in plant defense,12 cotton genotypes/varieties were evaluated for leaf area,leaf glanding,total soluble sugars,total soluble proteins,total phenolics,tannin and total flavonoids against fluctuating populations of whitefly,thrips and jassid under field conditions.Results:The population of these insects fluctuated during the growing seas on and remained above threshold level(whitefly>5,thrips>(8-10)f or jassid>1 per leaf)during late June and early July.Strong and negative association of whitefly(r=-0.825)and jassid(r=-0.929)with seed cotton yield was observed.Mean population of insects were the highest in Glandless-1 followed by NIA-82 and NIA-M30.NIAB-Kiran followed by NI AB-878 and Sadori were the most resistant,with the mean population of 1.41,1.60,1.66(whitefly);2.24,232,2.53(thrips)and 037,0.31,036(jassid),respectively.The resistant variety NIAB-Kiran showed less soluble sugars(8.54 mg.g^(-1)),soluble proteins(27.11 mg.g^(-1))and more phenolic(36.56 mg.g^(-1))and flavonoids(13.10mg.g^(-1))as compared with the susceptible check Glandless-1.Moreover,all insect populations were positively correlated with total soluble sugars and proteins.Whitefly populations exhibited negative response to leaf gossypol glands,total phenolics,tannins and flavonoids.The thrips and jassid populations had a significant and negative correlation with these four biochemical HPR traits.Conclusion:The ide ntified resistant resources and HPR traits can be deployed against sucking in sect pests'complex in future breeding programs of developing insect resistant cotton varieties.

Temporal and spatial changes in Bt toxin expression in Bt-transgenic poplar and insect resistance in field tests

Extensive planting of Bacillus thuringiensis （Bt）-transgenic plants economically benefits society; how-ever, the potential risk they pose is receiving increasing attention. This study used enzyme-linked immunosorbent assay and fluorescence quantitative PCR （RT-PCR） to monitor the temporal and spatial dynamics of the expression of Bt toxic protein in a forest of 6- to 8-year-old trees of transgenic insect-resistant poplar 741 for three consec- utive years. The enrichment, distribution, and degradation of Bt toxic protein and the influence of transgenic poplars on the targeted insect population, Hyphantria cunea, were investigated. The content of CrylAc toxic protein dynamically changed in transgenic poplar. During the annual growth cycle, the content initially increased, then decreased in the long and the short branches of the crown and in the root system, peaking in August. During the study, the protein did not accumulate overtime. The mRNA transcription of gene CrylAc was almost consistent with the level of the protein, but transcription peaked in July. In the transgenic and control forestland, microscale levels of the CrylAc toxic protein were detected from the soil, but increased accumulation was not observed with the planting year of transgenic poplar. Meanwhile, Bt was isolated and detected molecularly from the soil in the experimental forestland. A systematic investigation of the density of H. cunea in the experimental transgenic poplar forest indi- cated that transgenic Pb29 poplar could resist insects to a certain degree. At peak occurrence of the targeted insects, the density of H. cunea in the experimental forest was significantly lower than in the nontransgenic poplar forest.

Insect and Diseases Resistance in Tomato Entries

The study was conducted with 75 tomato entries at the farm of Olericulture Division, Horticulture Research Centre (HRC), Bangladesh Agricultural Research Institute (BARI), Gazipur, Bangladesh during the winter season of 2020-21 to evaluate insect and disease reaction. Among the various insect and diseases of tomatoes, the late blight, TYLCV, bacterial wilt infection and leaf miner, fruit borer infestation are most common in Bangladesh. The TYLCV infection was observed 0% to 27% infection, while 47 entries showed zero percent infection. The range of bacterial wilt infection was 0% to 10% and zero percent infection was observed in 62 entries. In case of leaf miner infestation and fruit borer infestation, the range was 0% to 43% and 0% to 10%, respectively. Considering tolerance to late blight, TYLCV, bacterial wilt infection and leaf miner, fruit borer infestation, fruit size, fruit shape, plant growth nature, cluster nature of fruit, type of fruit ten entries AVTO 1010, AVTO 1706, AVTO 1713, AVTO 1829, AVTO 1909, AVTO 1911, AVTO 1915, AVTO 1921, AVTO 1954 and SLA 011 were found zero percent late blight, TYLCV, bacterial wilt infection and leaf miner, fruit borer infestation. So, these ten entries can be selected for disease and insect tolerant tomato varieties development as well as developing disease and insect tolerant hybrid tomato varieties.

Research Progress in Molecular Mechanism of Broad-spectrum Disease Resistance in Rice

Yield loss caused by crop diseases seriously restricts global food security.Breeding disease-resistant varieties with resistance(R)genes is acknowledged as the most economical and effective way to control diseases.Therefore,exploring broad-spectrum resistance(BSR)genes to pathogens and clarifying the underlying mechanism are the basis for effective utilization of BSR resources and finding new approaches for disease control.In the past 20 years,significant progress has also been made in plant immune mechanism and remarkable achievements have been made in researches on broad-spectrum disease resistance of major grain crops,and the potential molecular mechanisms of some BSR have been revealed.Here,the advances of broad-spectrum disease resistance in rice(including the representative cloned BSR genes and their molecular mechanisms)were reviewed.In addition,the problems,opportunities and challenges encountered by BSR were analyzed,and the development of BSR research and its application in rice production were prospected.

Food Conditioning Affects Expression of Insect Resistance in Diploid Willows(Salix spp.)

The high energy quota and versatility of use make willows (Salix spp.) attractive as bioenergy crops. Insect defoliation constitutes a threat to the profitability of willow growers. Hitherto, the breeding for resistance against the main insect pests has been hampered by the fact that all known resistant willow clones are polyploids, and existing molecular breeding tools work most effectively for diploids. Here, we firstly report diploid willows highly resistant to the main insect defoliator, the leaf beetle (Phratora vulgatissima), offering new opportunities for breeding resistance. Leaf beetles exposed to three resistant clones (two S. purpurea one S. eriocephala) laid three to 27 times fewer eggs than females on a susceptible S. viminalis clone. Secondly, we show that beetles laid significantly more eggs on resistant clones if they were fed the susceptible clone prior to the oviposition monitoring test compared to when they prefed on resistant clones. Nevertheless, the differences observed between resistant and susceptible clones were pronounced in all cases. The food conditioning effect means that small differences in resistance among clones may be undetected.

Genetic Engineering of Tobacco with Double Resistance to Both Virus and Insect

Expression vector pE14 with double resistance to virus and insect was constructed by inserting CW-cp gene and Bt-toxin gene one by into T-DNA of the same binary vector pE3. Tobacco was then transformed with Agrbacterium tumefaciens (At)GV311-SE carrying PE14. Nopaline assay and PCR amplification confirmed that both CW-cp gene and Bt-toxin gene had been introduced into tobacco genome by T-DNA of PE3. Test attack with virus and demonstrated, in some of the transgenic plants, double resistance to both infection by CMV and damage by Manduca sexta.

Studies of Transgenic Hybrid Poplar 741 Carrying Two Insect-resistant Genes

Partially modified Bt Cry1Ac gene and the arrowhead proteinase inhibitor (API) gene were used to construct a plant transformation vector pBtiA and this construct was transferred into the genome of the hybrid poplar 741 [ Populus alba L.×( P. davidiana Dode+ P. simonii Carr.)× P. tomentosa Carr.] by Agrobacterium _ mediated transformation. Ten kanamycin resistant plants have been regenerated. Upon insect bioassay using Clostera anachoreta (Fabricius), three of the examined plants were demonstrated to be highly resistant to the testing insects. The mortality of insect larvae on one plant was higher than 90% in 6 days after infestation and the growth of the survival larvae were seriously inhibited. Results of PCR and Southern blot analysis indicated that both Bt Cry1Ac gene and API gene were integrated as a single copy into the genomes of these three plants when Cry1Ac gene fragment was used as the probe. Protein dot blot immunoassay and ELISA analysis revealed that at least the Cry1Ac protein was produced in these three transgenic plants and the expression levels were estimated to be approximately 0.015% of the leaf total soluble protein. This is the first report on insect resistant transgenic hybrid poplar 741 that expresses two insecticidal protein genes.

Evaluation and screening for resistance of some varieties(lines)to major diseases and insect pests of rice

During 1984-1988,2,231 varieties(lines)from International Rice Testing Program(IRTP)were evaluated and screened for resistance to riceblast(Bl),bacterial blight(BB),sheath blight

AlphaFold-guided redesign of a plant pectin methylesterase inhibitor for broad-spectrum disease resistance

Plant cell walls are a critical site where plants and pathogens continuously struggle for physiological domi-nance.Here we show that dynamic remodeling of pectin methylesterification of plant cell walls is a compo-nent of the physiological and co-evolutionary struggles between hosts and pathogens.A pectin methyles-terase(PsPME1)secreted by Phytophthora sojae decreases the degree of pectin methylesterification,thus synergizing with an endo-polygalacturonase(PsPG1)to weaken plant cell walls.To counter PsPME1-mediated susceptibility,a plant-derived pectin methylesterase inhibitor protein,GmPMl1,protects pectin to maintain a high methylesterification status.GmPMl1 protects plant cell walls from enzymatic degrada-tion by inhibiting both soybean and P.sojae pectin methylesterases during infection.However,constitutive expression of GmPMl1 disrupted the trade-off between host growth and defense responses.We therefore used AlphaFold structure tools to design a modified form of GmPMI1(GmPMI1R)that specifically targets and inhibits pectin methylesterases secreted from pathogens but notfrom plants.Transient expression of GmPMi1R enhanced plant resistance to oomycete and fungal pathogens.In summary,our work highlights the biochemical modification of the cell wall as an important focal point in the physiological and co-evolutionary conflict between hosts and microbes,providing an important proof of concept that Al-driven structure-based tools can accelerate the development of new strategies for plant protection.

Quantitative Trait Loci for Asian Corn Borer Resistance in Maize Population Mc37×Zi330

Asian corn borer, Ostrinia furnacalis （Guen6e）, is the most important pest of maize in China and Southeast Asia. The objective of this study was to understand the genetic basis for Asian corn borer resistance. The study included 162 F2：3 populations derived from Mc37 （resistant） × Zi330 （susceptible） and field data were collected in 2004 and 2005. A linkage map was constructed from 118 SSR markers. Combined quantitative trait loci （QTL） analysis combined across environments was performed by composite interval mapping method （CIM）. Four QTLs with additive effects were detected for resistance to Asian corn borer leaf feeding damage in chromosome bins 1.08, 2.04/05, 4.01, and 10.04. Three putative QTLs were detected for Asian corn borer stalk damage in chromosome bins 1.01, 2.05, and 9.01. Maize resistance to Asian corn borer and European corn borer, O. nubilalis （Hubner）, may share a common mechanism. Genes responsible for DIMBOA biosynthesis are in chromosome bin 4.01 and may increase the observed resistance to Asian corn borer leaf feeding.

Research Progress on the Mechanism of Plant Disease Resistance Regulated by NPR1

NPR1 is an intersection of different disease resistance pathways and plays a key role in systemic-acquired resistance and induced systemic resistance. NPR1 regulates constitutive resistance and disease resistance determined by resistant genes. In this paper, the mechanism of disease resistance of NPR1 genes and its analogs were described. The research status and progress of NPR1 transgenic crops were systematically discussed. The application prospect of NPR1 transgenic crops was also forecasted.