Mobile Host mRNAs Are Translated to Protein in the Associated Parasitic Plant Cuscuta campestris

Cuscuta spp. are obligate parasites that connect to host vascular tissue using a haustorium. In addition to water, nutrients, and metabolites, a large number of mRNAs are bidirectionally exchanged between Cuscuta spp. and their hosts. This trans-specific movement of mRNAs raises questions about whether these molecules function in the recipient species. To address the possibility that mobile mRNAs are ultimately translated, we built upon recent studies that demonstrate a role for transfer RNA (tRNA)-like structures (TLSs) in enhancing mRNA systemic movement. C. campestris was grown on Arabidopsis that expressed a β-glucuronidase (GUS) reporter transgene either alone or in GUS-tRNA fusions. Histochemical staining revealed localization in tissue of C. campestris grown on Arabidopsis with GUS-tRNA fusions, but not in C. campestris grown on Arabidopsis with GUS alone. This corresponded with detection of GUS transcripts in Cuscuta on Arabidopsis with GUS-tRNA, but not in C. campestris on Arabidopsis with GUS alone. Similar results were obtained with Arabidopsis host plants expressing the same constructs containing an endoplasmic reticulum localization signal. In C. campestris, GUS activity was localized in the companion cells or phloem parenchyma cells adjacent to sieve tubes. We conclude that host-derived GUS mRNAs are translated in C. campestris and that the TLS fusion enhances RNA mobility in the host-parasite interactions.

Structural deviations in secondary phloem of young shoots of Spiraea beauverdiana near magma volcanoes

We performed a comparative analysis of the internal structure of the secondary phloem of one, two and three-year-old stems of Spiraea beauverdiana growing in extreme conditions of solfataric fields of Golovnin Volcano caldera and Mendeleyev Volcano and in normal conditions. The combination of environmental factors in conditions of solfataric activity, such as high temperatures in the soil and in the near-surface air, as well as saturation with gases toxic to plants, rare elements accumulating in the nearby substrate, and lack of soil moisture, interfere with normal phellogen and cambium activity. Deviations from the normal structure involve changes in the following parameters of the internal structure of a year-old stem of S. beauverdiana. Secondary phloem parameters in the studied habitats are normal, except for the length of the segments of sieve tubes, the height of single-row rays, the length of parenchymal girder; these are shorter in S. beauverdiana stems from volcanos. At two and three years of age in volcanic conditions we see reduction in the width of the secondary phloem (both conductive and non-conductive) and the diameter of the segments of sieve tubes. In samples from Golovnin Volcano we see reduction in tangential diameter, while in the samples from Mendeleyev Volcano it’s the radial diameter. We also see reduction in the height of multiple-row rays. At that age we see changes in the structure of the radial parenchyma; namely, we find no double-row rays in samples from Golovnin Volcano caldera. One of the signs of impact of volcanic activity on the bark structure is development of non-specific anomalies in the internal structure of the S. beauverdiana bark, namely, in the outer bark, or in deeper levels, such as the secondary phloem. That causes sclerification and dilatation of parenchyma, and multiple layers in some tissues.

Detection, Identification and Molecular Characterization of a 16SrII-V subgroup Phytoplasma Associated with Nicotiana plumbaginifolia Viviani

Nicotiana plumbaginifolia Viviani or commonly known as curl-leaved tobacco is an annual herbaceous plant belonging to Solanaceae family. This plant is native to Mexico, South America, and parts of the Caribbean and has been reported to be present in Taiwan since 2006. In March 2021, N. plumbaginifolia Viviani found in Yunlin County, Taiwan was observed to have phyllody, virescence, and witches’-broom which is consistent with the disease symptoms caused by phytoplasma infection. Samples of the healthy and symptomatic plants were collected for analysis of the causal agent associated with the diseased N. plumbaginifolia Viviani. Under transmission electron microscopy, the phytoplasma-like pleomorphic bodies were found in the sieve tubes of the diseased plants. The 16S rRNA-based phylogenetic analysis and the iPhyClassifier-based virtual RFLP study demonstrated that the phytoplasma identified in this study can be classified into the 16SrII-V subgroup, which is similar to the peanut witches’-broom phytoplasma, a ‘Candidatus phytoplasma aurantifolia’-related strain. Further identification of SAP54/PHYL1 and SAP11 homologues in the phytoplasma explain the disease symptoms of phyllody, virescence, and witches’-broom observed in diseased N. plumbaginifolia Viviani. The discovery of new phytoplasma plant hosts has gained scientific importance in light of the attempt to unravel an efficient strategy to fight the rapid spread of this disease which poses threat to the agricultural sector and food security in Taiwan.

Evaluation of dsRNA delivery methods for targeting macrophage migration inhibitory factor MIF in RNAi-based aphid control

Macrophage migration inhibitory factors (MIFs) are multifunctional proteins regulating major processes in mammals, including activation of innate immune responses. In invertebrates, MIF proteins participate in the modulation of host immune responses when secreted by parasitic organisms, such as aphids. In this study, we assessed the possibility to use MIF genes as targets for RNA interference (RNAi)-based control of the grain aphid Sitobion avenae (Sa) on barley (Hordeum vulgare). When nymphs were fed on artificial diet containing double-stranded (ds)RNAs (SaMIF-dsRNAs) that target sequences of the three MIF genes SaMIF1, SaMIF2 and SaMIF3, they showed higher mortality rates and these rates correlated with reduced MIF transcript levels as compared to the aphids feeding on artificial diet containing a control dsRNA (GFP-dsRNA). Comparison of different feeding strategies showed that nymphs’ survival was not altered when they fed from barley seedlings sprayed with naked SaMIF-dsRNAs, suggesting they did not effectively take up dsRNA from the sieve tubes of these plants. Furthermore, aphids’ survival was also not affected when the nymphs fed on leaves supplied with dsRNA via basal cut ends of barley leaves. Consistent with this finding, the use of sieve tube-specific YFP-labeled Arabidopsis reporter lines confirmed that fluorescent 21 nt dsRNACy3, when supplied via petioles or spraying, co-localized with xylem structures, but not with phloem tissue. Our results suggest that MIF genes are a potential target for insect control and also imply that application of naked dsRNA to plants for aphid control is inefficient. More efforts should be put into the development of effective dsRNA formulations.

Evaluation of dsRNA delivery methods for targeting macrophage migration inhibitory factor MIF in RNAi-based aphid control

Macrophage migration inhibitory factors (MIF) are multifunctional proteins regulating major processes in mammals, including activation of innate immune responses. In invertebrates, MIF proteins participate in the modulation of host immune responses when secreted by parasitic organisms, such as aphids. In this study, we assessed the possibility to use MIF genes as targets for RNA interference (RNAi)-based control of the grain aphid Sitobion avenae (Sa) on barley (Hordeum vulgare). When nymphs were fed on artificial diet containing double-stranded (ds)RNAs (SaMIF-dsRNAs) that target sequences of the three MIF genes SaMIF1, SaMIF2 and SaMIF3, they showed higher mortality rates and these rates correlated with reduced MIF transcript levels as compared to the aphids feeding on artificial diet containing a control dsRNA (GFP-dsRNA). Comparison of different feeding strategies showed that nymphs’ survival was not altered when they fed from barley seedlings sprayed with SaMIF-dsRNAs, suggesting they did not effectively take up dsRNA from the sieve tubes of these plants. Furthermore, aphids’ survival was also not affected when the nymphs fed on leaves supplied with dsRNA via basal cut ends of barley leaves. Consistent with this finding, the use of sieve-tube-specific YFP-labeled Arabidopsis reporter lines confirmed that fluorescent 21 nt dsRNACy3 supplied via petioles co-localized with xylem structures, but not with phloem tissue. Our results suggest that MIF genes are a potential target for insect control and also imply that application of naked dsRNA to plants for aphid control is inefficient. More efforts should be put into the development of effective dsRNA formulations.

Identification of 16SrII-V Phytoplasma Associated with Mungbean Phyllody Disease in Taiwan

Mungbean (Vigna radiata (L.) R. Wilczek), an important legume crop in Asia, is primarily cultivated in the central-southern region of western Taiwan. In 2020, mungbean exhibiting typical phytoplasma-induced disease symptoms, such as witches’ broom, phyllody, virescence, and proliferation, was observed in Yunlin County, Taiwan. Moreover, the seeds harvested from diseased plants displayed premature germination. Transmission electron microscopy examination of leaf veins prepared from symptomatic mungbeans demonstrated that the occlusion of sieve tubes resulted from the accumulation of phytoplasma-like bodies in sieve elements along with filament-like structures in sieve pores. The association of phytoplasma in symptomatic mungbean was confirmed by PCR analyses of the 16S rRNA and immunodominant membrane protein genes. Further analyses of the 16S rRNA-based phylogenetic tree and the iPhyClassifier-based virtual RFLP study demonstrated that the phytoplasma-associated mungbean phyllody disease identified in this study belongs to the 16SrII-V subgroup. BLAST analysis and the phylogenetic analysis indicated that the SAP11-like protein identified in mungbean phyllody disease is identical to PnWB phytoplasma SAP11, which explains the witches’ broom phenotype observed in symptomatic mungbean. The results described in this report confirm that the 16SrII-V phytoplasma, a widely distributed phytoplasma associated with peanut witches’ broom disease in Taiwan, has also infected mungbean. This is not only the first instance of mungbean phyllody disease found in Taiwan, but also the first instance of mungbean phyllody disease causing by 16SrII-V subgroup phytoplasma.

Phloem development, growth markers, and sieve-tube longevity in two Neotropical trees

This study provides a detailed analysis of phloem anatomy, development, the formation of cell types, differentiation, and sieve-tube element’s longevity in two tropical arboreal species, Cedrela fissilis (Meliaceae, Rosid) and Citharexylum myrianthum (Verbenaceae, Asterid), growing in natural populations in the semi-deciduous Atlantic Rainforest. We periodically collected samples from the main stem at breast height (1.3 m), during both the dry and the wet seasons. Differences in the cells produced at these different seasons suggest that annual growth increments in the phloem are present in both species, marked by files of terminal narrow sieve-tube elements radially grouped in Cedrela fissilis, and in assemblages of narrow sieve tubes and axial parenchyma in Cytharexylum myrianthum, both appearing at the end of the wet season. In Cedrela fissilis, where fiber bands are present, each fiber band marks the end of the early phloem, acting as an indirect annual growth ring marker. Sieve-tube element longevity varied for both species from 4–26 months, a result similar to that obtained in temperate species.

Timeline of Leaf and Cambial Phenology in Relation to Development of Initial Conduits in Xylem and Phloem in Three Coexisting Sub-Mediterranean Deciduous Tree Species

It is unclear how the anticipated climate change will affect the timing of phenology of different tree organs/tissues and thus the whole-tree functioning. We examined the timing of leaf phenology and secondary growth in three coexisting deciduous tree species (Quercus pubescens Willd., Fraxinus ornus L. and Ostrya carpinifolia Scop) from a sub-Mediterranean region in 2019. In addition, we investigated the relationship between leaf and cambial phenology and the onset of the potential functioning of initial conduits, as determined by the completed differentiation process (vessels) or final size (sieve tubes). For this purpose, leaf development was monitored and the microcores of cambium and the youngest phloem and xylem increments were repeatedly collected at 7–10-day intervals during the growing season. The results revealed differences in the timing of leaf development and seasonal radial growth patterns in spring among the studied tree species, depending on wood porosity. We found that cambial cell production started in all cases in the first half of March. However, in ring-porous Q. pubescens and F. ornus, radial growth in the stem occurred more than a month before buds were swollen, whereas in diffuse-porous O. carpinifolia, these two events were detected at almost the same time. The end of cambial cell production occurred earliest in F. ornus (mid-July) and two weeks later also in the other two species. The widest initial earlywood vessels and early phloem sieve tubes were found in Q. pubescens, the narrowest initial earlywood vessels in O. carpinifolia and the narrowest early phloem sieve tubes in F. ornus. This indicates differences in the efficiency of conducting systems among the studied species. This novel approach of studying phloem phenology and anatomy in relation to leaf and xylem development contributes to a better understanding of how different tree species adapt their structure of secondary vascular tissues in response to environmental change.

Detection and molecular characterization of phytoplasma affecting vegetables in Eastern Visayas, Philippines

Phytoplasma-like diseases were observed affecting bitter gourd, Loofah, string bean, “Baguio” bean, cucumber, and tomato in Eastern Visayas, Philippines. The infected vegetables commonly show little leaf/witches’ broom symptoms. The study aimed to detect and confirm phytoplasmas presence in these vegetables through PCR and nest PCR assays using universal primers, electron microscopy, and 16srDNA sequence analysis. Loofah little leaf had the highest prevalence (50% of the surveyed farms), followed by bitter gourd (45%) and string beans (31%). The disease had an approximate mean incidence of 27% for bitter gourd, 38.0% for Loofah, and 42.5% for string bean, in farms where plants showed infections. Electron micrographs of bitter gourd and Loofah samples showed phytoplasma cells in the phloem sieve tubes. Nest PCR assays using R1 6F2n/R16R2 primer linked to phytoplasmal6srDNA amplified a ~1.25Kb band in the majority of DNA samples. rDNA sequence analysis using Blastn showed that phytoplasmas in bitter gourd, Loofah, and one cucumber samples shared 98-99% identity with Loofah’s reference gene phytoplasma clones. More than one phytoplasma strain infected the vegetables based on Rsai enzyme digestion and phylogenetic analysis.

MYB43 in Oilseed Rape (Brassica napus) Positively Regulates Vascular Lignification, Plant Morphology and Yield Potential but Negatively Affects Resistance to Sclerotinia sclerotiorum

Arabidopsis thaliana MYB43 (AtMYB43) is suggested to be involved in cell wall lignification. PtrMYB152, the Populus orthologue of AtMYB43, is a transcriptional activator of lignin biosynthesis and vessel wall deposition. In this research, MYB43 genes from Brassica napus (rapeseed) and its parental species B. rapa and B. oleracea were molecularly characterized, which were dominantly expressed in stem and other vascular organs and showed responsiveness to Sclerotinia sclerotiorum infection. The BnMYB43 family was silenced by RNAi, and the transgenic rapeseed lines showed retardation in growth and development with smaller organs, reduced lodging resistance, fewer silique number and lower yield potential. The thickness of the xylem layer decreased by 28%; the numbers of sclerenchymatous cells, vessels, interfascicular fibers, sieve tubes and pith cells in the whole cross section of the stem decreased by 28%, 59%, 48%, 34% and 21% in these lines, respectively. The contents of cellulose and lignin decreased by 17.49% and 16.21% respectively, while the pectin content increased by 71.92% in stems of RNAi lines. When inoculated with S. sclerotiorum, the lesion length was drastically decreased by 52.10% in the stems of transgenic plants compared with WT, implying great increase in disease resistance. Correspondingly, changes in the gene expression patterns of lignin biosynthesis, cellulose biosynthesis, pectin biosynthesis, cell cycle, SA- and JA-signals, and defensive pathways were in accordance with above phenotypic modifications. These results show that BnMYB43, being a growth-defense trade-off participant, positively regulates vascular lignification, plant morphology and yield potential, but negatively affects resistance to S. sclerotiorum. Moreover, this lignification activator influences cell biogenesis of both lignified and non-lignified tissues of the whole vascular organ.