Identification and functional characterization of the npc-2-like domain containing rust effector protein that suppresses cell death in plants.

The MD-2-related lipid-recognition (ML/Md-2) domain is a lipid/sterol-binding domain that are involved in sterol transfer and innate immunity in eukaryotes. Here we report a genome-wide survey of this family, identifying 84 genes in 30 fungi including plant pathogens. All the studied species were found to have varied ML numbers, and expansion of the family was observed in Rhizophagus irregularis (RI) with 33 genes. The molecular docking studies of these proteins with cholesterol derivatives indicate lipid-binding functional conservation across the animal and fungi kingdom. The phylogenetic studies among eukaryotic ML proteins showed that Puccinia ML members are more closely associated with animal (insect) npc2 proteins than other fungal ML members. One of the candidates from leaf rust fungus Puccinia triticina, Pt5643 was PCR amplified and further characterized using various studies such as qRT-PCR, subcellular localization studies, yeast functional complementation, signal peptide validation, and expression studies. The Pt5643 exhibits the highest expression on the 5th day post-infection (dpi). The confocal microscopy of Pt5643 in onion epidermal cells and N. benthamiana shows its location in the cytoplasm and nucleus. The functional complementation studies of Pt5643 in npc2 mutant yeast showed its functional similarity to the eukaryotic/yeast npc2 gene. Furthermore, the overexpression of Pt5643 also suppressed the BAX, NEP1, and H2O2-induced program cell death in Nicotiana species and yeast. Altogether the present study reports the novel function of ML domain proteins in plant fungal pathogens and their possible role as effector molecules in host defense manipulation.

Allantoin improves salinity tolerance in Arabidopsis and rice through synergid activation of abscisic acid and brassinosteroid biosynthesis.

Soil salinity stress is one of the major bottlenecks for crop production. Although, allantoin is known to be involved in nitrogen metabolism in plants, yet several reports in recent time indicate its involvement in various abiotic stress responses including salinity stress. However, the detail mechanism of allantoin involvement in salinity stress tolerance in plants is not studied well. Moreover, we demonstrated the role of exogenous application of allantoin as well as increased concentration of endogenous allantoin in rendering salinity tolerance in rice and Arabidopsis respectively, via., induction of abscisic acid (ABA) and brassinosteroid (BR) biosynthesis pathways. Exogenous application of allantoin (10 µM) provides  salt-tolerance to salt-sensitive rice genotype (IR-29). Transcriptomic data after exogenous supplementation of allantoin under salinity stress showed induction of ABA (OsNCED1) and BR (Oscytochrome P450) biosynthesis genes in IR-29. Further, the key gene of allantoin biosynthesis pathway i.e., urate oxidase of the halophytic species Oryza coarctata was also found to induce ABA and BR biosynthesis genes when over-expressed in transgenic Arabidopsis. Thus, indicating that ABA and BR biosynthesis pathways were involved in allantoin mediated salinity tolerance in both rice and Arabidopsis. Additionally, it has been found that several physio-chemical parameters such as biomass, Na+/K+ ratio, MDA, soluble sugar, proline, allantoin and chlorophyll contents were also associated with the allantoin-mediated salinity tolerance in urate oxidase overexpressed lines of Arabidopsis. These findings depicted the functional conservation of allantoin for salinity tolerance in both plant clades.

Intrinsically Disordered Kiwellin Protein-Like Effectors Target Plant Chloroplasts and are Extensively Present in Rust Fungi.

The effector proteins produced by plant pathogens are one of the essential components of host-pathogen interaction. Despite being important, most of the effector proteins remain unexplored due to the diversity in their primary sequence generated by the high selection pressure of the host immune system. However to maintain the primary function in the infection process, these effectors may tend to maintain their native protein fold to perform the corresponding biological function. In the present study, unannotated candidate secretory effector proteins of sixteen major plant fungal pathogens were analyzed to find the conserved known protein folds using homology, ab initio, and Alpha Fold/Rosetta Fold protein dimensional (3D) structure approaches. Several unannotated candidate effector proteins were found to match various known conserved protein families potentially involved in host defense manipulation in different plant pathogens. Surprisingly a large number of plant Kiwellin proteins fold like secretory proteins (> 100) were found in studied rust fungal pathogens. Many of them were predicted as potential effector proteins. Furthermore, template independent modelling using Alpha Fold/Rosetta Fold analysis and structural comparison of these candidates also predicted them to match with plant Kiwellin proteins. We also found plant Kiwellin matching proteins outside rusts including several non-pathogenic fungi suggesting the broad function of these proteins. One of the highest confidently modeled Kiwellin matching candidates effectors, Pstr_13960 (97.8%), from the Indian P. striiformis race Yr9 was characterized using overexpression, localization, and deletion studies in Nicotiana benthamiana. The Pstr_13960 suppressed the BAX-induced cell death and localized in the chloroplast. Furthermore, the expression of the Kiwellin matching region (Pst_13960_kiwi) alone suppressed the BAX-induced cell death in N. benthamiana despite the change of location to the cytoplasm and nucleus, suggesting the novel function of the Kiwellin core fold in rust fungi. Molecular docking showed that Pstr_13960 can interact with plant Chorismate mutases (CMs) using three loops conserved in plant and rust Kiwellins. Further analysis of Pstr_13960 showed to contain Intrinsically disordered regions (IDRs) in place of the N-terminal ß1/ß2 region found in plant Kiwellins suggesting the evolution of rust Kiwellins-like effectors (KLEs). Overall, this study reports the presence of a Kiwellin protein-like fold containing a novel effector protein family in rust fungi depicting a classical example of the evolution of effectors at the structure level as Kiwellin effectors show very low significant similarity to plant Kiwellin at the sequence level.

Genome-Wide Analysis of Four Pathotypes of Wheat Rust Pathogen (Puccinia graminis) Reveals Structural Variations and Diversifying Selection.

Diseases caused by Puccinia graminis are some of the most devastating diseases of wheat. Extensive genomic understanding of the pathogen has proven helpful not only in understanding host- pathogen interaction but also in finding appropriate control measures. In the present study, whole-genome sequencing of four diverse P. graminis pathotypes was performed to understand the genetic variation and evolution. An average of 63.5 Gb of data per pathotype with about 100× average genomic coverage was achieved with 100-base paired-end sequencing performed with Illumina Hiseq 1000. Genome structural annotations collectively predicted 9273 functional proteins including ~583 extracellular secreted proteins. Approximately 7.4% of the genes showed similarity with the PHI database which is suggestive of their significance in pathogenesis. Genome-wide analysis demonstrated pathotype 117-6 as likely distinct and descended through a different lineage. The 3-6% more SNPs in the regulatory regions and 154 genes under positive selection with their orthologs and under negative selection in the other three pathotypes further supported pathotype 117-6 to be highly diverse in nature. The genomic information generated in the present study could serve as an important source for comparative genomic studies across the genus Puccinia and lead to better rust management in wheat.

Effector Biology of Biotrophic Plant Fungal Pathogens: Current Advances and Future Prospects.

The interaction of fungal pathogens with their host requires a novel invading mechanism and the presence of various virulence-associated components responsible for promoting the infection. The small secretory proteins, explicitly known as effector proteins, are one of the prime mechanisms of host manipulation utilized by the pathogen to disarm the host. Several effector proteins are known to translocate from fungus to the plant cell for host manipulation. Many fungal effectors have been identified using genomic, transcriptomic, and bioinformatics approaches. Most of the effector proteins are devoid of any conserved signatures, and their prediction based on sequence homology is very challenging, therefore by combining the sequence consensus based upon machine learning features, multiple tools have also been developed for predicting apoplastic and cytoplasmic effectors. Various post-genomics approaches like transcriptomics of virulent isolates have also been utilized for identifying active consortia of effectors. Significant progress has been made in understanding biotrophic effectors; however, most of it is underway due to their complex interaction with host and complicated recognition and signaling networks. This review discusses advances, and challenges in effector identification and highlighted various features of the potential effector proteins and approaches for understanding their genetics and strategies for regulation.

Identification and characterization of Dicer-like genes in leaf rust pathogen (Puccinia triticina) of wheat.

Puccinia triticina (P. triticina) is one of the most devastating fungal pathogens of wheat which causes significant annual yield loss to the crop. Understanding the gene regulatory mechanism of the biotrophic pathogen is one of the important aspects of host-pathogen interaction studies. Dicer-like genes are considered as important mediators of RNAi-based gene regulation. In this study, we report the presence of three Dicer-like genes (Pt-DCL1, Pt-DCL2, Pt-DCL3) in P. triticina genome identified through computational and biological analyses. Quantitative real-time PCR studies revealed an increase in the expression of these genes in germinating spore stages. Heterologous expression combined with mass spectrometry analysis of Pt-DCL2 confirmed the presence of a canonical Dicer-like gene in P. triticina. Phylogenetic analysis of the Pt-DCLs with the Dicer-like proteins from other organisms showed a distinct cluster of rust pathogens from the order Pucciniales. The results indicated a species-specific duplication of Dicer-like genes within the wheat rust pathogens. This study, for the first time, reports the presence of Dicer-dependent RNAi pathway in P. triticina that may play a role in gene regulatory mechanism of the pathogen during its development. Our study serves as a vital source of information for further RNAi-based molecular studies for better understanding and management of the wheat leaf rust disease.

Transcriptional dynamics of Zn-accumulation in developing kernels of maize reveals important Zn-uptake mechanisms.

In the present study, transcriptomic analysis of 10-days old baby kernels of two contrasting maize genotypes, namely VQL-2 (high kernel Zn accumulator) and CM-145 (low kernel Zn accumulator), under low- and optimum- soil Zn conditions generated 1948 differentially expressed transcripts. Among these, 666 and 437 transcripts were up-regulated and down-regulated respectively in VQL-2; whereas, 437 and 408 transcripts were up-regulated and down-regulated respectively in CM-145. Remarkably, 135 transcription factors and 77 known Zn transporters expressed differentially. By comparing the transcripts differentially expressed between the optimum-Zn and low-Zn libraries of the contrasting genotypes, we identified 21,986 and 26,871 SNPs, respectively. Similarly, 6810 and 8192 InDels were found between optimum- and low-Zn growing conditions, respectively. Further, 21 differentially expressed genes were co-localized with already known QTLs associated with Zn uptake, such as qZn10, CQZnK9-1 and YNZnK6. These findings will be useful to develop high Zn-accumulator maize through marker-assisted breeding in future.

Discovery and profiling of small RNAs from Puccinia triticina by deep sequencing and identification of their potential targets in wheat.

Cross-kingdom RNAi is a well-documented phenomenon where sRNAs generated by host and pathogens may govern resistance or susceptible phenotypes during host-pathogen interaction. With the first example of the direct involvement of fungal generated sRNAs in virulence of plant pathogenic fungi Botrytis cinerea and recently from Puccinia striiformis f. sp. tritici, we attempted to identify sRNAs in Puccinia triticina (P. triticina). Four sRNA libraries were prepared and sequenced using Illumina sequencing technology and a total of ~ 1-1.28 million potential sRNAs and two microRNA-like small RNA (mil-RNAs) candidates were identified. Computational prediction of targets using a common set of sRNAs and P. triticina mil-RNAs (pt-mil-RNAs) within P. triticina and wheat revealed the majority of the targets as repetitive elements in P. triticina whereas in wheat, the target genes were identified to be involved in many biological processes including defense-related pathways. We found 9 receptor-like kinases (RLKs) and 14 target genes of each related to reactive oxygen species (ROS) pathway and transcription factors respectively, including significant numbers of target genes from various other categories. Expression analysis of twenty selected sRNAs, targeting host genes pertaining to ROS related, disease resistance, metabolic processes, transporter, apoptotic inhibitor, and transcription factors along with two pt-mil-RNAs by qRT-PCR showed distinct patterns of expression of the sRNAs in urediniospore-specific libraries. In this study, for the first time, we report identification of novel sRNAs identified in P. triticina including two pt-mil-RNAs that may play an important role in biotrophic growth and pathogenicity.

Status and Prospects of Next Generation Sequencing Technologies in Crop Plants.

The history of DNA sequencing dates back to 1970s. During this period the two first generation nucleotide sequencing techniques were developed. Subsequently the Sanger's dideoxy method of sequencing gained popularity over Maxam and Gilbert's chemical method of sequencing. However, in the last decade, we have observed revolutionary changes in DNA sequencing technologies leading to the emergence of next-generation sequencing (NGS) techniques. NGS technologies have enhanced the throughput and speed of sequencing combined with bringing down the overall cost of the process over a time. The major applications of NGS technologies being genome sequencing and resequencing, transcriptomics, metagenomics in relation to plant-microbe interactions, exon and genome capturing, development of molecular markers and evolutionary studies. In this review, we present a broader picture of evolution of NGS tools, its various applications in crop plants, and future prospects of the technology for crop improvement.

Dissection of genomic features and variations of three pathotypes of Puccinia striiformis through whole genome sequencing.

Stripe rust of wheat, caused by Puccinia striiformis f. sp. tritici, is one of the important diseases of wheat. We used NGS technologies to generate a draft genome sequence of two highly virulent (46S 119 and 31) and a least virulent (K) pathotypes of P. striiformis from the Indian subcontinent. We generated ~24,000-32,000 sequence contigs (N50;7.4-9.2 kb), which accounted for ~86X-105X sequence depth coverage with an estimated genome size of these pathotypes ranging from 66.2-70.2 Mb. A genome-wide analysis revealed that pathotype 46S 119 might be highly evolved among the three pathotypes in terms of year of detection and prevalence. SNP analysis revealed that ~47% of the gene sets are affected by nonsynonymous mutations. The extracellular secreted (ES) proteins presumably are well conserved among the three pathotypes, and perhaps purifying selection has an important role in differentiating pathotype 46S 119 from pathotypes K and 31. In the present study, we decoded the genomes of three pathotypes, with 81% of the total annotated genes being successfully assigned functional roles. Besides the identification of secretory genes, genes essential for pathogen-host interactions shall prove this study as a huge genomic resource for the management of this disease using host resistance.

Draft Genome of the Wheat Rust Pathogen (Puccinia triticina) Unravels Genome-Wide Structural Variations during Evolution.

Leaf rust is one of the most important diseases of wheat and is caused by Puccinia triticina, a highly variable rust pathogen prevalent worldwide. Decoding the genome of this pathogen will help in unraveling the molecular basis of its evolution and in the identification of genes responsible for its various biological functions. We generated high quality draft genome sequences (approximately 100- 106 Mb) of two races of P. triticina; the variable and virulent Race77 and the old, avirulent Race106. The genomes of races 77 and 106 had 33X and 27X coverage, respectively. We predicted 27678 and 26384 genes, with average lengths of 1,129 and 1,086 bases in races 77 and 106, respectively and found that the genomes consisted of 37.49% and 39.99% repetitive sequences. Genome wide comparative analysis revealed that Race77 differs substantially from Race106 with regard to segmental duplication (SD), repeat element, and SNP/InDel characteristics. Comparative analyses showed that Race 77 is a recent, highly variable and adapted Race compared with Race106. Further sequence analyses of 13 additional pathotypes of Race77 clearly differentiated the recent, active and virulent, from the older pathotypes. Average densities of 2.4 SNPs and 0.32 InDels per kb were obtained for all P. triticina pathotypes. Secretome analysis demonstrated that Race77 has more virulence factors than Race 106, which may be responsible for the greater degree of adaptation of this pathogen. We also found that genes under greater selection pressure were conserved in the genomes of both races, and may affect functions crucial for the higher levels of virulence factors in Race77. This study provides insights into the genome structure, genome organization, molecular basis of variation, and pathogenicity of P. triticina The genome sequence data generated in this study have been submitted to public domain databases and will be an important resource for comparative genomics studies of the more than 4000 existing Puccinia species.

The TATA-box sequence in the basal promoter contributes to determining light-dependent gene expression in plants.

A prototype 13-bp TATA-box sequence, TCACTATATATAG, was mutated at each nucleotide position and examined for its function in the core promoter. Specific nucleotides in the first TATA, the second TATA, as well as the flanking sequences influenced promoter function in transient transformation of tobacco (Nicotiana tabacum var Petit Havana) leaves. The effect of a given mutation on reporter gene expression in light versus dark was variable and sometimes contrasting. Some mutations, like T(7) or A(8)-->C or G, completely inactivated the expression of the minimal promoter in light but not in dark. In general, the sequence requirement for dark expression was less stringent than that for light expression. The selective effect of TATA-box mutations on light versus dark expression was exerted on core promoter function in the chromatin-integrated state also. Even in the presence of an upstream light response activator element, TATA-box mutations influenced modulation of the promoter by light. An A at the eighth position was specifically involved in the red light response of the promoter. Selectivity in gene expression was associated with a high level of transcript initiation from a site that was not active in the dark. Nuclear proteins from dark- and light-grown seedlings showed that the sequence variation within the TATA-box governs the formation of alternative transcriptional complexes. The experiments give direct evidence for the role of a core TATA-box sequence in determining the level as well as selectivity of gene expression in plants.

Analysis of polarity in the expression from a multifactorial bidirectional promoter designed for high-level expression of transgenes in plants.

A synthetic bidirectional expression module was constructed by placing a computationally designed minimal promoter sequence on the 5' and 3' sides of a transcription activation module. The activation of transcription from the unidirectional and bidirectional promoters constructed from the same sequence elements was evaluated by using the reporter genes gusA and gfp. The analysis based on transient and stable transformation of tobacco showed that the artificially designed multifactorial activation module activated transcription simultaneously to comparable levels in both the directions. The transcription activation module responded to elicitors like salicylic acid, NaCl and IAA in the forward as well as reverse directions. The concentration of the elicitor required for highest gene activation was similar for the two directions in case of the three activators. The kinetics of time of induction was similar in the two directions for salicylic acid and NaCl. In the case of IAA, the transcription activation was faster in the reverse direction. The results show that constitutive and chemically inducible bidirectional promoters can be deployed for predictable simultaneous regulation of two genes for genetic engineering in plants.

A variety of synergistic and antagonistic interactions mediated by cis-acting DNA motifs regulate gene expression in plant cells and modulate stability of the transcription complex formed on a basal promoter.

Several synthetic promoters containing a variety of commonly found cis-acting DNA sequence motifs were constructed to study the motif-motif and motif-protein interactions involved in gene expression in plants. Transient expression of the reporter gene gusA in tobacco leaves was used to demonstrate that several sequence elements can be arranged upstream of a basal promoter to function synergistically in enhancing gene expression. A cis-acting DNA motif could function as an activator by itself as well as a synergizing activator in the presence of other homologous as well as heterologous motifs in the neighbourhood. The function of a complex promoter comprising several activation motifs was arrested nearly completely in vivo, following titration with any one of the motifs. The results suggested a hierarchical assembly of several motif-binding factors, leading to the stabilization of the transcriptional complex formed on the TATA-box.