One-Step Reverse-Transcription Loop-Mediated Isothermal Amplification (RT-LAMP) Assay for the Detection of Canna Yellow Streak Virus.

Canna yellow streak virus (CaYSV) is a potyvirus that causes severe damage to the ornamental plant canna in the United Kingdom and Brazil. Here, we identified CaYSV in China by isolating total RNA from an infected plant, amplifying the virus genome segments, and cloning and sequencing the amplicons. After assembly, the full-length genome of the virus was obtained and uploaded to the NCBI database. Phylogenetic analysis results showed that the Guizhou isolate (OL546222) was most closely related to the KS isolate (MG545919.1). Virus detection is essential for virus disease control but the subclinical infection of CaYSV on canna in its early development increases the difficulty of CaYSV diagnosis. The goal of this study was to develop an efficient method for detection of CaYSV. We designed the primers, optimized the reaction conditions, and finally established a one-step reverse-transcription loop-mediated isothermal amplification (RT-LAMP) method. The product of RT-LAMP can be analyzed by both agarose gel electrophoresis and visible color change. The established one-step RT-LAMP assay showed high specificity and sensitivity in detecting CaYSV. This RT-LAMP method was also applied in analysis of 61 field samples collected from Guizhou and Jiangsu Provinces. The results showed that the infection rates of CaYSV on canna samples from these two provinces were very high (63 and 96% respectively).

A chromosome-scale Gastrodia elata genome and large-scale comparative genomic analysis indicate convergent evolution by gene loss in mycoheterotrophic and parasitic plants.

Mycoheterotrophic and parasitic plants are heterotrophic and parasitize on fungi and plants, respectively, to obtain nutrients. Large-scale comparative genomics analysis has not been conducted in mycoheterotrophic or parasitic plants or between these two groups of parasites. We assembled a chromosome-level genome of the fully mycoheterotrophic plant Gastrodia elata (Orchidaceae) and performed comparative genomic analyses on the genomes of G. elata and four orchids (initial mycoheterotrophs), three parasitic plants (Cuscuta australis, Striga asiatica, and Sapria himalayana), and 36 autotrophs from various angiosperm lineages. It was found that while in the hemiparasite S. asiatica and initial mycoheterotrophic orchids, approximately 4-5% of the conserved orthogroups were lost, the fully heterotrophic G. elata and C. australis both lost approximately 10% of the conserved orthogroups, indicating that increased heterotrophy is positively associated with gene loss. Importantly, many genes that are essential for autotrophs, including those involved in photosynthesis, the circadian clock, flowering time regulation, immunity, nutrient uptake, and root and leaf development, were convergently lost in both G. elata and C. australis. The high-quality genome of G. elata will facilitate future studies on the physiology, ecology, and evolution of mycoheterotrophic plants, and our findings highlight the critical role of gene loss in the evolution of plants with heterotrophic lifestyles.

Herbivory-induced systemic signals are likely to be evolutionarily conserved in euphyllophytes.

Herbivory-induced systemic signaling has been demonstrated in monocots and dicots, and is essential for plant defense against insects. However, the nature and evolution of herbivory-induced systemic signals remain unclear. Grafting is widely used for studying systemic signaling; however, grafting between dicot plants from different families is difficult, and grafting is impossible for monocots. In this study, we took advantage of dodder's extraordinary capability of parasitizing various plant species. Field dodder (Cuscuta campestris) was employed to connect pairs of species that are phylogenetically very distant, ranging from fern to monocot and dicot plants, and so determine whether interplant signaling occurs after simulated herbivory. It was found that simulated herbivory-induced systemic signals can be transferred by dodder between a monocot and a dicot plant and even between a fern and a dicot plant, and the plants that received the systemic signals all exhibited elevated defenses. Thus, we inferred that the herbivory-induced systemic signals are likely to be evolutionarily well conserved among vascular plants. Importantly, we also demonstrate that the jasmonate pathway is probably an ancient regulator of the biosynthesis and/or transport of systemic signals in vascular plants. These findings provide new insight into the nature and evolution of systemic signaling.

Stem parasitic plant Cuscuta australis (dodder) transfers herbivory-induced signals among plants.

Cuscuta spp. (i.e., dodders) are stem parasites that naturally graft to their host plants to extract water and nutrients; multiple adjacent hosts are often parasitized by one or more Cuscuta plants simultaneously, forming connected plant clusters. Metabolites, proteins, and mRNAs are known to be transferred from hosts to Cuscuta, and Cuscuta bridges even facilitate host-to-host virus movement. Whether Cuscuta bridges transmit ecologically meaningful signals remains unknown. Here we show that, when host plants are connected by Cuscuta bridges, systemic herbivory signals are transmitted from attacked plants to unattacked plants, as revealed by the large transcriptomic changes in the attacked local leaves, undamaged systemic leaves of the attacked plants, and leaves of unattacked but connected hosts. The interplant signaling is largely dependent on the jasmonic acid pathway of the damaged local plants, and can be found among conspecific or heterospecific hosts of different families. Importantly, herbivore attack of one host plant elevates defensive metabolites in the other systemic Cuscuta bridge-connected hosts, resulting in enhanced resistance against insects even in several consecutively Cuscuta-connected host plants over long distances (> 100 cm). By facilitating plant-to-plant signaling, Cuscuta provides an information-based means of countering the resource-based fitness costs to their hosts.

MYC2, MYC3, and MYC4 function additively in wounding-induced jasmonic acid biosynthesis and catabolism.

Jasmonic acid (JA) plays a critical role in plant defenses against insects and necrotrophic fungi. Wounding or lepidopteran insect feeding rapidly induces a burst of JA in plants, which usually reaches peak values within 1 to 2 h. The induced JA is converted to JA-Ile and perceived by the COI1-JAZ co-receptor, leading to activation of the transcription factors MYC2 and its homologs, which further induce JA-responsive genes. Although much is known about JA biosynthesis and catabolism enzymes and JA signaling, how JA biosynthesis and catabolism are regulated remain unclear. Here, we show that in Arabidopsis thaliana MYC2 functions additively with MYC3 and MYC4 to regulate wounding-induced JA accumulation by directly binding to the promoters of genes function in JA biosynthesis and catabolism to promote their transcription. MYC2 also controls the transcription of JAV1 and JAM1, which are key factors controlling JA biosynthesis and catabolism, respectively. In addition, we also found that MYC2 could bind to the MYC2 promoter and self-inhibit its own expression. This work illustrates the central role of MYC2/3/4 in controlling wounding-induced JA accumulation by regulating the transcription of genes involved in JA biosynthesis and catabolism.

Comparative analysis of alfalfa (Medicago sativa L.) leaf transcriptomes reveals genotype-specific salt tolerance mechanisms.

BACKGROUND: Soil salinity is an important factor affecting growth, development, and productivity of almost all land plants, including the forage crop alfalfa (Medicago sativa). However, little is known about how alfalfa responds and adapts to salt stress, particularly among different salt-tolerant cultivars. RESULTS: Among seven alfalfa cultivars, we found that Zhongmu-1 (ZM) is relatively salt-tolerant and Xingjiang Daye (XJ) is salt-sensitive. Compared to XJ, ZM showed slower growth under low-salt conditions, but exhibited stronger tolerance to salt stress. RNA-seq analysis revealed 2237 and 1125 differentially expressed genes (DEGs) between ZM and XJ in the presence and absence of salt stress, among which many genes are involved in stress-related pathways. After salt treatment, compared with the controls, the number of DEGs in XJ (19373) was about four times of that in ZM (4833). We also detected specific differential gene expression patterns: In response to salt stress, compared with XJ, ZM maintained relatively more stable expression levels of genes related to the ROS and Ca2+ pathways, phytohormone biosynthesis, and Na+/K+ transport. Notably, several salt resistance-associated genes always showed greater levels of expression in ZM than in XJ, including a transcription factor. Consistent with the suppression of plant growth resulting from salt stress, the expression of numerous photosynthesis- and growth hormone-related genes decreased more dramatically in XJ than in ZM. By contrast, the expression levels of photosynthetic genes were lower in ZM under low-salt conditions. CONCLUSIONS: Compared with XJ, ZM is a salt-tolerant alfalfa cultivar possessing specific regulatory mechanisms conferring exceptional salt tolerance, likely by maintaining high transcript levels of abiotic and biotic stress resistance-related genes. Our results suggest that maintaining this specific physiological status and/or plant adaptation to salt stress most likely arises by inhibition of plant growth in ZM through plant hormone interactions. This study identifies new candidate genes that may regulate alfalfa tolerance to salt stress and increases the understanding of the genetic basis for salt tolerance.

Elevated CO2 differentially affects tobacco and rice defense against lepidopteran larvae via the jasmonic acid signaling pathway.

Atmospheric CO2 levels are rapidly increasing due to human activities. However, the effects of elevated CO2 (ECO2 ) on plant defense against insects and the underlying mechanisms remain poorly understood. Here we show that ECO2 increased the photosynthetic rates and the biomass of tobacco and rice plants, and the chewing lepidopteran insects Spodoptera litura and Mythimna separata gained less and more mass on tobacco and rice plants, respectively. Consistently, under ECO2 , the levels of jasmonic acid (JA), the main phytohormone controlling plant defense against these lepidopteran insects, as well as the main defense-related metabolites, were increased and decreased in insect-damaged tobacco and rice plants. Importantly, bioassays and quantification of defense-related metabolites in tobacco and rice silenced in JA biosynthesis and perception indicate that ECO2 changes plant resistance mainly by affecting the JA pathway. We further demonstrate that the defensive metabolites, but not total N or protein, are the main factors contributing to the altered defense levels under ECO2 . This study illustrates that ECO2 changes the interplay between plants and insects, and we propose that crops should be studied for their resistance to the major pests under ECO2 to predict the impact of ECO2 on future agroecosystems.

Cloning and characterization of chromosomal markers in alfalfa (Medicago sativa L.).

Eleven tandemly repetitive sequences were identified from a Cot-1 library by FISH and sequence analysis of alfalfa (Medicago sativa). Five repetitive sequences (MsCR-1, MsCR-2, MsCR-3, MsCR-4, and MsCR-5) were centromeric or pericentromeric, of which three were satellite DNAs and two were minisatellite DNAs. Monomers of 144, 148, and 168 bp were identified in MsCR-1, MsCR-2, and MsCR-3, respectively, while 15 and 39 bp monomers were identified in MsCR-4 and MsCR-5, respectively. Three repetitive sequences were characterized as subtelomeric; one repetitive sequence, MsTR-1, had a 184 bp monomer, and two repetitive sequences had fragments of 204 and 327 bp. Sequence analysis revealed homology (70-80 %) between MsTR-1 and a highly repeated sequence (C300) isolated from M. ssp. caerulea. Three identified repetitive sequences produced hybridization signals at multiple sites in a few of the chromosomes; one repetitive sequence was identified as the E180 satellite DNA previously isolated from M. sativa, while the other 163 and 227 bp fragments had distinct sequences. Physical mapping of the repetitive sequences with double-target FISH revealed different patterns. Thus, nine novel tandemly repetitive sequences that can be adopted as distinct chromosome markers in alfalfa were identified in this study. Furthermore, the chromosome distribution of each sequence was well described. Though significant chromosome variations were detected within and between cultivars, a molecular karyotype of alfalfa was suggested with the chromosome markers we identified. Therefore, these novel chromosome markers will still be a powerful tool for genome composition analysis, phylogenetic studies, and breeding applications.

Between-Plant Signaling.

Parasitic plants use a special organ, the haustorium, to attach to and penetrate host tissues, forming phloem and/or xylem fusion with the host vascular systems. Across this haustorium-host interface, not only water and nutrients are extracted from the host by the parasitic plant, but also secondary metabolites, messenger RNAs, noncoding RNAs, proteins, and systemic signals are transported between the parasite and host and even among different hosts connected by a parasite. Furthermore, mycorrhizal fungi can form common mycelial networks (CMNs) that simultaneously interconnect multiple plants. Increasing lines of evidence suggest that CMNs can function as conduits, transferring stress-related systemic signals between plants. Between-plant signaling mediated by haustoria and CMNs likely has a profound impact on plant interactions with other organisms and adaptation to environmental factors. Here, we summarize the findings regarding between-plant transfer of biomolecules and systemic signals and the current understanding of the physiological and ecological implications of between-plant signaling.

Characterization of alien chromosomes in backcross derivatives of Triticum aestivum × Elymus rectisetus hybrids by using molecular markers and sequential multicolor FISH/GISH.

Wild Triticeae grasses serve as important gene pools for forage and cereal crops. Based on DNA sequences of genome-specific RAPD markers, sequence-tagged site (STS) markers specific for W and Y genomes have been obtained. Coupling with the use of genomic in situ hybridization, these STS markers enabled the identification of the W- and Y-genome chromosomes in backcross derivatives from hybrids of bread wheat Triticum aestivum L. (2n=42; AABBDD) and Elymus rectisetus (Nees in Lehm.) Á. Löve & Connor (2n=42; StStWWYY). The detection of six different alien chromosomes in five of these derivatives was ascertained by quantitative PCR of STS markers, simple sequence repeat markers, rDNA genes, and (or) multicolor florescence in situ hybridization. Disomic addition line 4687 (2n=44) has the full complement of 42 wheat chromosomes and a pair of 1Y chromosomes that carry genes for resistance to tan spot (caused by Pyrenophora tritici-repentis (Died.) Drechs.) and Stagonospora nodorum blotch (caused by Stagonospora nodorum (Berk.) Castellani and Germano). The disomic addition line 4162 has a pair of 1St chromosomes and 21 pairs of wheat chromosomes. Lines 4319 and 5899 are two triple substitution lines (2n=42) having the same chromosome composition, with 2A, 4B, and 6D of wheat substituted by one pair of W- and two pairs of St-genome chromosomes. Line 4434 is a substitution-addition line (2n=44) that has the same W- and St-genome chromosomes substituting 2A, 4B, and 6D of wheat as in lines 4319 and 5899 but differs by having an additional pair of Y-genome chromosome, which is not the 1Y as in line 4687. The production and identification of these alien cytogenetic stocks may help locate and isolate genes for useful agronomic traits.

Comparative genomics of orobanchaceous species with different parasitic lifestyles reveals the origin and stepwise evolution of plant parasitism.

Orobanchaceae is the largest family of parasitic plants, containing autotrophic and parasitic plants with all degrees of parasitism. This makes it by far the best family for studying the origin and evolution of plant parasitism. Here we provide three high-quality genomes of orobanchaceous plants, the autotrophic Lindenbergia luchunensis and the holoparasitic plants Phelipanche aegyptiaca and Orobanche cumana. Phylogenomic analysis of these three genomes together with those previously published and the transcriptomes of other orobanchaceous species created a robust phylogenetic framework for Orobanchaceae. We found that an ancient whole-genome duplication (WGD; about 73.48 million years ago), which occurred earlier than the origin of Orobanchaceae, might have contributed to the emergence of parasitism. However, no WGD events occurred in any lineage of orobanchaceous parasites except for Striga after divergence from their autotrophic common ancestor, suggesting that, in contrast with previous speculations, WGD is not associated with the emergence of holoparasitism. We detected evident convergent gene loss in all parasites within Orobanchaceae and between Orobanchaceae and dodder Cuscuta australis. The gene families in the orobanchaceous parasites showed a clear pattern of recent gains and expansions. The expanded gene families are enriched in functions related to the development of the haustorium, suggesting that recent gene family expansions may have facilitated the adaptation of orobanchaceous parasites to different hosts. This study illustrates a stepwise pattern in the evolution of parasitism in the orobanchaceous parasites and will facilitate future studies on parasitism and the control of parasitic plants in agriculture.

Salt-tolerant and -sensitive alfalfa (Medicago sativa) cultivars have large variations in defense responses to the lepidopteran insect Spodoptera litura under normal and salt stress condition.

In nature, plants are often exposed to multiple stress factors at the same time. Yet, little is known about how plants modulate their physiology to counteract simultaneous abiotic and biotic stresses, such as soil salinity and insect herbivory. In this study, insect performance bioassays, phytohormone measurements, quantification of transcripts, and protein determination were employed to study the phenotypic variations of two alfalfa (Medicago sativa) cultivars in response to insect Spodoptera litura feeding under normal and salt stress condition. When being cultivated in normal soil, the salt-tolerant alfalfa cultivar Zhongmu-1 exhibited lower insect resistance than did the salt-sensitive cultivar Xinjiang Daye. Under salinity stress, the defense responses of Xinjiang Daye were repressed, whereas Zhongmu-1 did not show changes in resistance levels. It is likely that salinity influenced the resistance of Xinjiang Daye through suppressing the accumulation of jasmonic acid-isoleucine (JA-Ile), which is the bioactive hormone inducing herbivore defense responses, leading to attenuated trypsin proteinase inhibitor (TPI) activity. Furthermore, exogenous ABA supplementation suppressed the insect herbivory-induced JA/JA-Ile accumulation and levels of JAR1 (jasmonate resistant 1) and TPI, and further decreased the resistance of Xinjiang Daye, whereas Zhongmu-1 showed very little response to the increased ABA level. We propose a mechanism, in which high levels of abscisic acid induced by salt treatment may affect the expression levels of JAR1 and consequently decrease JA-Ile accumulation and thus partly suppress the defense of Xinjiang Daye against insects under salt stress. This study provides new insight into the mechanism by which alfalfa responds to concurrent abiotic and biotic stresses.