Towards defining the core Saccharum microbiome: input from five genotypes.

BACKGROUND: Plant microbiome and its manipulation inaugurate a new era for plant biotechnology with the potential to benefit sustainable crop production. Here, we used the large-scale 16S rDNA sequencing analysis to unravel the dynamic, structure, and composition of exophytic and endophytic microbial communities in two hybrid commercial cultivars of sugarcane (R570 and SP80-3280), two cultivated genotypes (Saccharum officinarum and Saccharum barberi) and one wild species (Saccharum spontaneum). RESULTS: Our analysis identified 1372 amplicon sequence variants (ASVs). The microbial communities' profiles are grouped by two, root and bulk soils and stem and leave when these four components are compared. However, PCoA-based data supports that endophytes and epiphytes communities form distinct groups, revealing an active host-derived mechanism to select the resident microbiota. A strong genotype-influence on the assembly of microbial communities in Saccharum ssp. is documented. A total of 220 ASVs persisted across plant cultivars and species. The ubiquitous bacteria are two potential beneficial bacteria, Acinetobacter ssp., and Serratia symbiotica. CONCLUSIONS: The results presented support the existence of common and cultivar-specific ASVs in two commercial hybrids, two cultivated canes and one species of Saccharum across tissues (leaves, stems, and roots). Also, evidence is provided that under the experimental conditions described here, each genotype bears its microbial community with little impact from the soil conditions, except in the root system. It remains to be demonstrated which aspect, genotype, environment or both, has the most significant impact on the microbial selection in sugarcane fields.

Subtilase activity in intrusive cells mediates haustorium maturation in parasitic plants.

Parasitic plants that infect crops are devastating to agriculture throughout the world. These parasites develop a unique inducible organ called the haustorium that connects the vascular systems of the parasite and host to establish a flow of water and nutrients. Upon contact with the host, the haustorial epidermal cells at the interface with the host differentiate into specific cells called intrusive cells that grow endophytically toward the host vasculature. Following this, some of the intrusive cells re-differentiate to form a xylem bridge (XB) that connects the vasculatures of the parasite and host. Despite the prominent role of intrusive cells in host infection, the molecular mechanisms mediating parasitism in the intrusive cells remain poorly understood. In this study, we investigated differential gene expression in the intrusive cells of the facultative parasite Phtheirospermum japonicum in the family Orobanchaceae by RNA-sequencing of laser-microdissected haustoria. We then used promoter analyses to identify genes that are specifically induced in intrusive cells, and promoter fusions with genes encoding fluorescent proteins to develop intrusive cell-specific markers. Four of the identified intrusive cell-specific genes encode subtilisin-like serine proteases (SBTs), whose biological functions in parasitic plants are unknown. Expression of SBT inhibitors in intrusive cells inhibited both intrusive cell and XB development and reduced auxin response levels adjacent to the area of XB development. Therefore, we propose that subtilase activity plays an important role in haustorium development in P. japonicum.

Ethylene signaling mediates host invasion by parasitic plants.

Parasitic plants form a specialized organ, a haustorium, to invade host tissues and acquire water and nutrients. To understand the molecular mechanism of haustorium development, we performed a forward genetics screening to isolate mutants exhibiting haustorial defects in the model parasitic plant Phtheirospermum japonicum. We isolated two mutants that show prolonged and sometimes aberrant meristematic activity in the haustorium apex, resulting in severe defects on host invasion. Whole-genome sequencing revealed that the two mutants respectively have point mutations in homologs of ETHYLENE RESPONSE 1 (ETR1) and ETHYLENE INSENSITIVE 2 (EIN2), signaling components in response to the gaseous phytohormone ethylene. Application of the ethylene signaling inhibitors also caused similar haustorial defects, indicating that ethylene signaling regulates cell proliferation and differentiation of parasite cells. Genetic disruption of host ethylene production also perturbs parasite invasion. We propose that parasitic plants use ethylene as a signal to invade host roots.

Genome Sequence of Striga asiatica Provides Insight into the Evolution of Plant Parasitism.

Parasitic plants in the genus Striga, commonly known as witchweeds, cause major crop losses in sub-Saharan Africa and pose a threat to agriculture worldwide. An understanding of Striga parasite biology, which could lead to agricultural solutions, has been hampered by the lack of genome information. Here, we report the draft genome sequence of Striga asiatica with 34,577 predicted protein-coding genes, which reflects gene family contractions and expansions that are consistent with a three-phase model of parasitic plant genome evolution. Striga seeds germinate in response to host-derived strigolactones (SLs) and then develop a specialized penetration structure, the haustorium, to invade the host root. A family of SL receptors has undergone a striking expansion, suggesting a molecular basis for the evolution of broad host range among Striga spp. We found that genes involved in lateral root development in non-parasitic model species are coordinately induced during haustorium development in Striga, suggesting a pathway that was partly co-opted during the evolution of the haustorium. In addition, we found evidence for horizontal transfer of host genes as well as retrotransposons, indicating gene flow to S. asiatica from hosts. Our results provide valuable insights into the evolution of parasitism and a key resource for the future development of Striga control strategies.

A new glance on root-to-shoot in vivo zinc transport and time-dependent physiological effects of ZnSO4 and ZnO nanoparticles on plants.

Understanding nanoparticle root uptake and root-to-shoot transport might contribute to the use of nanotechnology in plant nutrition. This study performed time resolved experiments to probe Zn uptake, biotransformation and physiological effects on Phaseolus vulgaris (L.). Plants roots were exposed to ZnO nanoparticles (40 and 300 nm) dispersions and ZnSO4(aq) (100 and 1000 mg Zn L-1) for 48 h. Near edge X-ray absorption spectroscopy showed that 40 nm ZnO was more easily dissolved by roots than 300 nm ZnO. It also showed that in the leaves Zn was found as a mixture Zn3(PO4)2 and Zn-histidine complex. X-ray fluorescence spectroscopy showed that root-to-shoot Zn-translocation presented a decreasing gradient of concentration and velocity, it seems radial Zn movement occurs simultaneously to the axial xylem transport. Below 100 mg Zn L-1, the lower stem tissue section served as a buffer preventing Zn from reaching the leaves. Conversely, it was not observed for 1000 mg Zn L-1 ZnSO4(aq). Transcriptional analysis of genes encoding metal carriers indicated higher expression levels of tonoplast-localized transporters, suggesting that the mechanism trend to accumulate Zn in the lower tissues may be associated with an enhanced of Zn compartmentalization in vacuoles. The photosynthetic rate, transpiration, and water conductance were impaired by treatments.

Quinone oxidoreductase 2 is involved in haustorium development of the parasitic plant Phtheirospermum japonicum.

The family Orobanchaceae includes many parasitic plant species. Parasitic plants invade host vascular tissues and form organs called haustoria, which are used to obtain water and nutrients. Haustorium formation is initiated by host-derived chemicals including quinones and flavonoids. Two types of quinone oxidoreductase (QR) are involved in signal transduction leading to haustorium formation; QR1 mediates single-electron transfers and QR2 mediates 2-electron transfers. In the facultative parasite Triphysaria versicolor, QR1 is involved in haustorium induction signaling, while this role is played by QR2 in the model plant Phtheirospermum japonicum. Our results suggest that there is functional diversification in haustorium signaling molecules among different species of the Orobanchaceae.

Haustorium Induction Assay of the Parasitic Plant Phtheirospermum japonicum.

Phtheirospermum japonicum is a facultative root parasitic plant in the Orobanchaceae family used as a model parasitic plant. Facultative root parasites form an invasive organ called haustorium on the lateral parts of their roots. To functionally characterize parasitic abilities, quantification of haustorium numbers is required. However, this task is quite laborious and time consuming. Here we describe an efficient protocol to induce haustorium in vitro by haustorium-inducing chemicals and host root exudate treatments in P. japonicum.

Local Auxin Biosynthesis Mediated by a YUCCA Flavin Monooxygenase Regulates Haustorium Development in the Parasitic Plant Phtheirospermum japonicum.

Parasitic plants in the Orobanchaceae cause serious agricultural problems worldwide. Parasitic plants develop a multicellular infectious organ called a haustorium after recognition of host-released signals. To understand the molecular events associated with host signal perception and haustorium development, we identified differentially regulated genes expressed during early haustorium development in the facultative parasite Phtheirospermum japonicum using a de novo assembled transcriptome and a customized microarray. Among the genes that were upregulated during early haustorium development, we identified YUC3, which encodes a functional YUCCA (YUC) flavin monooxygenase involved in auxin biosynthesis. YUC3 was specifically expressed in the epidermal cells around the host contact site at an early time point in haustorium formation. The spatio-temporal expression patterns of YUC3 coincided with those of the auxin response marker DR5, suggesting generation of auxin response maxima at the haustorium apex. Roots transformed with YUC3 knockdown constructs formed haustoria less frequently than nontransgenic roots. Moreover, ectopic expression of YUC3 at the root epidermal cells induced the formation of haustorium-like structures in transgenic P. japonicum roots. Our results suggest that expression of the auxin biosynthesis gene YUC3 at the epidermal cells near the contact site plays a pivotal role in haustorium formation in the root parasitic plant P. japonicum.

Agrobacterium rhizogenes-mediated transformation of the parasitic plant Phtheirospermum japonicum.

BACKGROUND: Plants within the Orobanchaceae are an agriculturally important group of parasites that attack economically important crops to obtain water and nutrients from their hosts. Despite their agricultural importance, molecular mechanisms of the parasitism are poorly understood. METHODOLOGY/PRINCIPAL FINDINGS: We developed transient and stable transformation systems for Phtheirospermum japonicum, a facultative parasitic plant in the Orobanchaceae. The transformation protocol was established by a combination of sonication and acetosyringone treatments using the hairy-root-inducing bacterium, Agrobacterium rhizogenes and young seedlings. Transgenic hairy roots of P. japonicum were obtained from cotyledons 2 to 3 weeks after A. rhizogenes inoculation. The presence and the expression of transgenes in P. japonicum were verified by genomic PCR, Southern blot and RT-PCR methods. Transgenic roots derived from A. rhizogenes-mediated transformation were able to develop haustoria on rice and maize roots. Transgenic roots also formed apparently competent haustoria in response to 2,6-dimethoxy-1,4-benzoquinone (DMBQ), a haustorium-inducing chemical. Using this system, we introduced a reporter gene with a Cyclin B1 promoter into P. japonicum, and visualized cell division during haustorium formation. CONCLUSIONS: We provide an easy and efficient method for hairy-root transformation of P. japonicum. Transgenic marker analysis revealed that cell divisions during haustorium development occur 24 h after DMBQ treatment. The protocols described here will allow functional analysis of genes involved in plant parasitism.

A full-length enriched cDNA library and expressed sequence tag analysis of the parasitic weed, Striga hermonthica.

BACKGROUND: The obligate parasitic plant witchweed (Striga hermonthica) infects major cereal crops such as sorghum, maize, and millet, and is the most devastating weed pest in Africa. An understanding of the nature of its parasitism would contribute to the development of more sophisticated management methods. However, the molecular and genomic resources currently available for the study of S. hermonthica are limited. RESULTS: We constructed a full-length enriched cDNA library of S. hermonthica, sequenced 37,710 clones from the library, and obtained 67,814 expressed sequence tag (EST) sequences. The ESTs were assembled into 17,317 unigenes that included 10,319 contigs and 6,818 singletons. The S. hermonthica unigene dataset was subjected to a comparative analysis with other plant genomes or ESTs. Approximately 80% of the unigenes have homologs in other dicotyledonous plants including Arabidopsis, poplar, and grape. We found that 589 unigenes are conserved in the hemiparasitic Triphysaria species but not in other plant species. These are good candidates for genes specifically involved in plant parasitism. Furthermore, we found 1,445 putative simple sequence repeats (SSRs) in the S. hermonthica unigene dataset. We tested 64 pairs of PCR primers flanking the SSRs to develop genetic markers for the detection of polymorphisms. Most primer sets amplified polymorphicbands from individual plants collected at a single location, indicating high genetic diversity in S. hermonthica. We selected 10 primer pairs to analyze S. hermonthica harvested in the field from different host species and geographic locations. A clustering analysis suggests that genetic distances are not correlated with host specificity. CONCLUSIONS: Our data provide the first extensive set of molecular resources for studying S. hermonthica, and include EST sequences, a comparative analysis with other plant genomes, and useful genetic markers. All the data are stored in a web-based database and freely available. These resources will be useful for genome annotation, gene discovery, functional analysis, molecular breeding, epidemiological studies, and studies of plant evolution.