Physiological, Nutritional, and Biochemical Bases of Corn Resistance to Foliage-Feeding Fall Armyworm

Three corn (Zea mays) germplasm lines [i.e., Ab24E (susceptible control), Mp708 (resistant control), and a locally selected partial inbred line FAW7050 (resistant)] were examined for Spodoptera frugiperda (J.E. Smith; Lepidoptera: Noctuidae) resistance. Nutritional [i.e., total protein content, amino acids, glucose, total nonstructural carbohydrates (TNC), protein to TNC (P/C) ratios] and biochemical (i.e., peroxidase and lipoxygenase 3) properties in the seedlings of these corn lines were examined to categorize resistance mechanisms to S. frugiperda. Physiological changes in photosynthetic rates also were examined in an attempt to explain nutritional and biochemical dynamics among corn germplasm lines and between insect-infested and noninfested corn plants within a germplasm line. Results indicated that S. frugiperda larvae survived better and developed faster in susceptible Ab24E than in resistant FAW7050 or Mp708. The three germplasm lines differed in resistance mechanisms to S. frugiperda, and the observed patterns of resistance were probably collective results of the P/C ratio and defensive proteins. That is, the susceptibility of Ab24E to S. frugiperda was due to a high P/C ratio and a low level of induced defensive compounds in response to insect herbivory, while the resistance of FAW7050 resulted from elevated defensive proteins following insect herbivory, low P/C ratio, and elevated defensive proteins in Mp708 contributed to its resistance to S. frugiperda. The elevated protein amounts in resistant Mp708 and FAW7050 following S. frugiperda injury were likely due to greater conversion of photosynthates to defensive proteins following the greater photosynthetic rates in these entries. Greater photosynthetic capacity in Mp708 and FAW7050 also led to higher amino acid and glucose contents in these two lines. Neither amino acid nor lipoxygenase 3 played a critical role in corn resistance to S. frugiperda. However, high inducibility of peroxidase may be an indicator of S. frugiperda susceptibility as observed elsewhere.     

Characterization of Erysiphe necator-Responsive Genes in Chinese Wild Vitis quinquangularis

Powdery mildew (PM), caused by fungus Erysiphe necator, is one of the most devastating diseases of grapevine. To better understand grapevine-PM interaction and provide candidate resources for grapevine breeding, a suppression subtractive hybridization (SSH) cDNA library was constructed from E. necator-infected leaves of a resistant Chinese wild Vitis quinquangularis clone “Shang-24”. A total of 492 high quality expressed sequence tags (ESTs) were obtained and assembled into 266 unigenes. Gene ontology (GO) analysis indicated that 188 unigenes could be assigned with at least one GO term in the biological process category, and 176 in the molecular function category. Sequence analysis showed that a large number of these genes were homologous to those involved in defense responses. Genes involved in metabolism, photosynthesis, transport and signal transduction were also enriched in the library. Expression analysis of 13 selected genes by qRT-PCR revealed that most were induced more quickly and intensely in the resistant material “Shang-24” than in the sensitive V. pseudoreticulata clone “Hunan-1” by E. necator infection. The ESTs reported here provide new clues to understand the disease-resistance mechanism in Chinese wild grapevine species and may enable us to investigate E. necator-responsive genes involved in PM resistance in grapevine germplasm.     

GhTBL34 Is Associated with Verticillium Wilt Resistance in Cotton

Verticillium wilt (VW) is a typical fungal disease affecting the yield and quality of cotton. The Trichome Birefringence-Like protein (TBL) is an acetyltransferase involved in the acetylation process of cell wall polysaccharides. Up to now, there are no reports on whether the TBL gene is related to disease resistance in cotton. In this study, we cloned a cotton TBL34 gene located in the confidence interval of a major VW resistance quantitative trait loci and demonstrated its relationship with VW resistance in cotton. Analyzing the sequence variations in resistant and susceptible accessions detected two elite alleles GhTBL34-2 and GhTBL34-3, mainly presented in resistant cotton lines whose disease index was significantly lower than that of susceptible lines carrying the allele GhTBL34-1. Comparing the TBL34 protein sequences showed that two amino acid differences in the TBL (PMR5N) domain changed the susceptible allele GhTBL34-1 into the resistant allele GhTBL34-2 (GhTBL34-3). Expression analysis showed that the TBL34 was obviously up-regulated by infection of Verticillium dahliae and exogenous treatment of ethylene (ET), and salicylic acid (SA) and jasmonate (JA) in cotton. VIGS experiments demonstrated that silencing of TBL34 reduced VW resistance in cotton. We deduced that the TBL34 gene mediating acetylation of cell wall polysaccharides might be involved in the regulation of resistance to VW in cotton.     

Changes in Bacterial Endophyte Community Following Aspergillus flavus Infection in Resistant and Susceptible Maize Kernels

Aspergillus flavus (A. flavus)-mediated aflatoxin contamination in maize is a major global economic and health concern. As A. flavus is an opportunistic seed pathogen, the identification of factors contributing to kernel resistance will be of great importance in the development of novel mitigation strategies. Using V3–V4 bacterial rRNA sequencing and seeds of A. flavus-resistant maize breeding lines TZAR102 and MI82 and a susceptible line, SC212, we investigated kernel-specific changes in bacterial endophytes during infection. A total of 81 bacterial genera belonging to 10 phyla were detected. Bacteria belonging to the phylum Tenericutes comprised 86–99% of the detected phyla, followed by Proteobacteria (14%) and others (<5%) that changed with treatments and/or genotypes. Higher basal levels (without infection) of Streptomyces and Microbacterium in TZAR102 and increases in the abundance of Stenotrophomonas and Sphingomonas in MI82 following infection may suggest their role in resistance. Functional profiling of bacteria using 16S rRNA sequencing data revealed the presence of bacteria associated with the production of putative type II polyketides and sesquiterpenoids in the resistant vs. susceptible lines. Future characterization of endophytes predicted to possess antifungal/ anti-aflatoxigenic properties will aid in their development as effective biocontrol agents or microbiome markers for maize aflatoxin resistance.     

Leaf-to-Whole Plant Spread Bioassay for Pepper and Ralstonia solanacearum Interaction Determines Inheritance of Resistance to Bacterial Wilt for Further Breeding

Bacterial wilt (BW) disease from Ralstonia solanacearum is a serious disease and causes severe yield losses in chili peppers worldwide. Resistant cultivar breeding is the most effective in controlling BW. Thus, a simple and reliable evaluation method is required to assess disease severity and to investigate the inheritance of resistance for further breeding programs. Here, we developed a reliable leaf-to-whole plant spread bioassay for evaluating BW disease and then, using this, determined the inheritance of resistance to R. solanacearum in peppers. Capsicum annuum ‘MC4′ displayed a completely resistant response with fewer disease symptoms, a low level of bacterial cell growth, and significant up-regulations of defense genes in infected leaves compared to those in susceptible ‘Subicho’. We also observed the spreading of wilt symptoms from the leaves to the whole susceptible plant, which denotes the normal BW wilt symptoms, similar to the drenching method. Through this, we optimized the evaluation method of the resistance to BW. Additionally, we performed genetic analysis for resistance inheritance. The parents, F₁ and 90 F₂ progenies, were evaluated, and the two major complementary genes involved in the BW resistance trait were confirmed. These could provide an accurate evaluation to improve resistant pepper breeding efficiency against BW.     

Hormonal Responses to Susceptible,Intermediate, and Resistant Interactions in the Brassica napus–Leptosphaeria maculans Pathosystem

Hormone signaling plays a pivotal role in plant–microbe interactions. There are three major phytohormones in plant defense: salicylic acid (SA), jasmonic acid (JA), and ethylene (ET). The activation and trade-off of signaling between these three hormones likely determines the strength of plant defense in response to pathogens. Here, we describe the allocation of hormonal signaling in Brassica napus against the fungal pathogen Leptosphaeria maculans. Three B. napus genotypes (Westar, Surpass400, and 01-23-2-1) were inoculated with two L. maculans isolates (H75 8-1 and H77 7-2), subsequently exhibiting three levels of resistance: susceptible, intermediate, and resistant. Quantitative analyses suggest that the early activation of some SA-responsive genes, including WRKY70 and NPR1, contribute to an effective defense against L. maculans. The co-expression among factors responding to SA/ET/JA was also observed in the late stage of infection. The results of conjugated SA measurement also support that early SA activation plays a crucial role in durable resistance. Our results demonstrate the relationship between the onset patterns of certain hormone regulators and the effectiveness of the defense of B. napus against L. maculans.     

Hairpin RNA Targeting Multiple Viral Genes Confers Strong Resistance to Rice Black-Streaked Dwarf Virus

Rice black-streaked dwarf virus (RBSDV) belongs to the genus Fijivirus in the family of Reoviridae and causes severe yield loss in rice-producing areas in Asia. RNA silencing, as a natural defence mechanism against plant viruses, has been successfully exploited for engineering virus resistance in plants, including rice. In this study, we generated transgenic rice lines harbouring a hairpin RNA (hpRNA) construct targeting four RBSDV genes, S1, S2, S6 and S10, encoding the RNA-dependent RNA polymerase, the putative core protein, the RNA silencing suppressor and the outer capsid protein, respectively. Both field nursery and artificial inoculation assays of three generations of the transgenic lines showed that they had strong resistance to RBSDV infection. The RBSDV resistance in the segregating transgenic populations correlated perfectly with the presence of the hpRNA transgene. Furthermore, the hpRNA transgene was expressed in the highly resistant transgenic lines, giving rise to abundant levels of 21–24 nt small interfering RNA (siRNA). By small RNA deep sequencing, the RBSDV-resistant transgenic lines detected siRNAs from all four viral gene sequences in the hpRNA transgene, indicating that the whole chimeric fusion sequence can be efficiently processed by Dicer into siRNAs. Taken together, our results suggest that long hpRNA targeting multiple viral genes can be used to generate stable and durable virus resistance in rice, as well as other plant species.     

Functional Identification of Serine Hydroxymethyltransferase as a Key Gene Involved in Lysostaphin Resistance and Virulence Potential of Staphylococcus aureus Strains

Gaining an insight into the mechanism underlying antimicrobial-resistance development in Staphylococcus aureus is crucial for identifying effective antimicrobials. We isolated S. aureus sequence type 72 from a patient in whom the S. aureus infection was highly resistant to various antibiotics and lysostaphin, but no known resistance mechanisms could explain the mechanism of lysostaphin resistance. Genome-sequencing followed by subtractive and functional genomics revealed that serine hydroxymethyltransferase (glyA or shmT gene) plays a key role in lysostaphin resistance. Serine hydroxymethyltransferase (SHMT) is indispensable for the one-carbon metabolism of serine/glycine interconversion and is linked to folate metabolism. Functional studies revealed the involvement of SHMT in lysostaphin resistance, as ΔshmT was susceptible to the lysostaphin, while complementation of the knockout expressing shmT restored resistance against lysostaphin. In addition, the ΔshmT showed reduced virulence under in vitro (mammalian cell lines infection) and in vivo (wax-worm infection) models. The SHMT inhibitor, serine hydroxymethyltransferase inhibitor 1 (SHIN1), protected the 50% of the wax-worm infected with wild type S. aureus. These results suggest SHMT is relevant to the extreme susceptibility to lysostaphin and the host immune system. Thus, the current study established that SHMT plays a key role in lysostaphin resistance development and in determining the virulence potential of multiple drug-resistant S. aureus.     

Genome-Wide Analysis of Ribosomal Protein GhRPS6 and Its Role in Cotton Verticillium Wilt Resistance

Verticillium wilt is threatening the world’s cotton production. The pathogenic fungus Verticillium dahliae can survive in the soil in the form of microsclerotia for a long time, colonize through the root of cotton, and invade into vascular bundles, causing yellowing and wilting of cotton leaves, and in serious cases, leading to plant death. Breeding resistant varieties is the most economical and effective method to control Verticillium wilt. In previous studies, proteomic analysis was carried out on different cotton varieties inoculated with V. dahliae strain Vd080. It was found that GhRPS6 was phosphorylated after inoculation, and the phosphorylation level in resistant cultivars was 1.5 times than that in susceptible cultivars. In this study, knockdown of GhRPS6 expression results in the reduction of SA and JA content, and suppresses a series of defensive response, enhancing cotton plants susceptibility to V. dahliae. Overexpression in Arabidopsis thaliana transgenic plants was found to be more resistant to V. dahliae. Further, serines at 237 and 240 were mutated to phenylalanine, respectively and jointly. The transgenic Arabidopsis plants demonstrated that seri-237 compromised the plant resistance to V. dahliae. Subcellular localization in Nicotiana benthamiana showed that GhRPS6 was localized in the nucleus. Additionally, the pathogen inoculation and phosphorylation site mutation did not change its localization. These results indicate that GhRPS6 is a potential molecular target for improving resistance to Verticillium wilt in cotton. This lays a foundation for breeding disease-resistant varieties.