Identification and characterization of TMV-induced volatile signals in Nicotiana benthamiana: evidence for JA/ET defense pathway priming in congeneric neighbors via airborne (E)-2-octenal.

Plants release a mixture of volatile compounds when subjects to environmental stress, allowing them to transmit information to neighboring plants. Here, we find that Nicotiana benthamiana plants infected with tobacco mosaic virus (TMV) induces defense responses in neighboring congeners. Analytical screening of volatiles from N. benthamiana at 7 days post inoculation (dpi) using an optimized SPME-GC-MS method showed that TMV triggers the release of several volatiles, such as (E)-2-octenal, 6-methyl-5-hepten-2-one, and geranylacetone. Exposure to (E)-2-octenal enhances the resistance of N. benthamiana plants to TMV and triggers the immune system with upregulation of pathogenesis-related genes, such as NbPR1a, NbPR1b, NbPR2, and NbNPR1, which are related to TMV resistance. Furthermore, (E)-2-octenal upregulates jasmonic acid (JA) that levels up to 400-fold in recipient N. benthamiana plants and significantly affects the expression pattern of key genes in the JA/ET signaling pathway, such as NbMYC2, NbERF1, and NbPDF1.2, while the salicylic acid (SA) level is not significantly affected. Our results show for the first time that the volatile (E)-2-octenal primes the JA/ET pathway and then activates immune responses, ultimately leading to enhanced TMV resistance in adjacent N. benthamiana plants. These findings provide new insights into the role of airborne compounds in virus-induced interplant interactions.

Carbon nanosol-induced assemblage of a plant-beneficial microbiome consortium.

Carbon nanosol (CNS) is a carbon-based nanomaterial that promotes plant growth; however, its functional mechanisms and effects on the microbiome are not fully understood. Here, we explored the effects of CNS on the relationship between the soil, endophytic microbiomes and plant productivity. CNS treatment increased the fresh biomass of tobacco (Nicotiana tabacum L.) plants by 27.4% ± 9.9%. Amplicon sequencing analysis showed that the CNS treatment significantly affected the composition and diversity of the microbial communities in multiple ecological niches associated with tobacco, especially the bulk soil and stem endophytic microbiome. Furthermore, the application of CNS resulted in enhanced network connectivity and stability of the microbial communities in different niches, particularly in the soil, implying a strengthening of certain microbial interactions. Certain potentially growth-promoting root endophytic bacteria were more abundant under the CNS treatment. In addition, CNS increased the abundance of some endophytic microbial functional genes known to enhance plant growth, such as those associated with nutrient metabolism and the plant hormone biosynthesis pathways. We isolated two bacterial strains (Sphingopyxis sp. and Novosphingobium sp.) that were enriched under CNS treatment, and they were confirmed to promote tobacco plant growth in vitro. These results suggested that CNS might, at least in part, promote plant growth by enriching beneficial bacteria in the microbiome.

Genome-wide identification and analysis of the invertase gene family in tobacco (Nicotiana tabacum) reveals NtNINV10 participating the sugar metabolism.

Sucrose (Suc) is directly associated with plant growth and development as well as tolerance to various stresses. Invertase (INV) enzymes played important role in sucrose metabolism by irreversibly catalyzing Suc degradation. However, genome-wide identification and function of individual members of the INV gene family in Nicotiana tabacum have not been conducted. In this report, 36 non-redundant NtINV family members were identified in Nicotiana tabacum including 20 alkaline/neutral INV genes (NtNINV1-20), 4 vacuolar INV genes (NtVINV1-4), and 12 cell wall INV isoforms (NtCWINV1-12). A comprehensive analysis based on the biochemical characteristics, the exon-intron structures, the chromosomal location and the evolutionary analysis revealed the conservation and the divergence of NtINVs. For the evolution of the NtINV gene, fragment duplication and purification selection were major factors. Besides, our analysis revealed that NtINV could be regulated by miRNAs and cis-regulatory elements of transcription factors associated with multiple stress responses. In addition, 3D structure analysis has provided evidence for the differentiation between the NINV and VINV. The expression patterns in diverse tissues and under various stresses were investigated, and qRT-PCR experiments were conducted to confirm the expression patterns. Results revealed that changes in NtNINV10 expression level were induced by leaf development, drought and salinity stresses. Further examination revealed that the NtNINV10-GFP fusion protein was located in the cell membrane. Furthermore, inhibition of the expression of NtNINV10 gene decreased the glucose and fructose in tobacco leaves. Overall, we have identified possible NtINV genes functioned in leaf development and tolerance to environmental stresses in tobacco. These findings provide a better understanding of the NtINV gene family and establish the basis for future research.

Functional identification of the DHN melanin synthesis gene cluster and its role in UV-C tolerance in citrus postharvest pathogenic fungus Penicillium digitatum.

The filamentous fungus Penicillium digitatum brings out great losses in citrus fruits by causing citrus green mold disease during the postharvest period. Previously, we obtained a T-DNA insertion mutant N2130 of P. digitatum, which produced albino conidia. To understand the role of green-grey conidial pigment in P. digitatum, we identified the insertion site and deeply explored the 1,8-dihydroxynaphtsalene (DHN)-melanin synthesis gene cluster in this phytopathogen. In this study, we deleted five genes in P. digitatum, PdPksP, PdAbr1, PdArp1, PdArp2, and PdAyg1, and the experiments were further performed on phenotype analyses, including pigmentation, UV-C tolerance, virulence, growth rate, conidiation, stress (osmotic-, oxidative-, cell wall disturbing-, and high temperature-) tolerance, fungicide resistance, and conidial hydrophobicity. The results showed that the five deletion mutants (ΔPdPksP, ΔPdAbr1, ΔPdArp1, ΔPdArp2 and ΔPdAyg1) produced albino, brownish, brown, reddish-brown, and Yellowish green conidia, respectively. In addition, the survival colony forming units (CFUs) of the deletion mutants, under the treatment of UV-C radiation (261.4 mJ/cm2), were 0.3- to 0.6-fold of those surviving in wild-type strain N1. Moreover, after 522.8 mJ/cm2-UV-C-irradiation on conidia, the deletion mutants showed a larger decrease in pathogenicity on Valencia Orange fruits compared with strain N1. However, there were no significant differences among other phenotypes tested in this study. Collectively, our research reported the DHN-melanin synthesis pathway in P. digitatum for the first time, and revealed that DHN-melanin is important for P. digitatum to tolerate UV-C irradiation.

Genome-Wide Identification and Analysis of the Class III Peroxidase Gene Family in Tobacco (Nicotiana tabacum).

Class III peroxidases (PODs) are plant-specific enzymes that play significant roles in plant physiological processes and stress responses. However, a comprehensive analysis of the POD gene family in tobacco has not yet been conducted. In this study, 210 non-redundant POD gene members (NtPODs) were identified in tobacco (Nicotiana tabacum) and distributed unevenly throughout 24 tobacco chromosomes. Phylogenetic analysis clustered these genes into six subgroups (I-VI). Gene structure and motif analyses showed the structural and functional diversity among the subgroups. Segmental duplication and purifying selection were the main factors affecting NtPOD gene evolution. Our analyses also suggested that NtPODs might be regulated by miRNAs and cis-acting regulatory elements of transcription factors that are involved in various biological processes. In addition, the expression patterns in different tissues and under various stress treatments were investigated. The results showed that the majority of NtPODs had tissue-specific expression patterns and may be involved in many biotic and abiotic responses. qRT-PCR analyses of different tissues and stress treatments were performed to verify transcriptome patterns. Expression of a green fluorescent protein-NtPOD fusion confirmed the plasma membrane localization of NtPOD121 and NtPOD4. Furthermore, 3D structures provided evidences of membrane-bound peroxidase. These findings provide useful information to better understand the evolution of the NtPOD gene family and lay the foundation for further studies on POD gene function in tobacco.

Comprehensive analysis of the carboxylesterase gene reveals that NtCXE22 regulates axillary bud growth through strigolactone metabolism in tobacco.

Carboxylesterases (CXE) are a class of hydrolytic enzymes with α/ß-folding domains that play a vital role in plant growth, development, stress response, and activation of herbicide-active substances. In this study, 49 Nicotiana tabacum L. CXE genes (NtCXEs) were identified using a sequence homology search. The basic characteristics, phylogenetic evolution, gene structure, subcellular location, promoter cis-elements, and gene expression patterns of the CXE family were systematically analyzed. RNA-seq data and quantitative real-time PCR showed that the expression level of CXEs was associated with various stressors and hormones; gene expression levels were significantly different among the eight tissues examined and at different developmental periods. As a new class of hormones, strigolactones (SLs) are released from the roots of plants and can control the germination of axillary buds.NtCXE7, NtCXE9, NtCXE22, and NtCXE24 were homologous to Arabidopsis SLs hydrolase AtCXE15, and changes in their expression levels were induced by topping and by GR24 (a synthetic analogue of strigolactone). Further examination revealed that NtCXE22-mutant (ntcxe22) plants generated by CRISPR-Cas9 technology had shorter bud outgrowth with lower SLs content. Validation of NtCXE22 was also performed in NtCCD8-OE plants (with fewer axillary buds) and in ntccd8 mutant plants (with more axillary buds). The results suggest that NtCXE22 may act as an efficient SLs hydrolase and affects axillary bud development, thereby providing a feasible method for manipulating endogenous SLs in crops and ornamental plants.