How plants manage pathogen infection.

To combat microbial pathogens, plants have evolved specific immune responses that can be divided into three essential steps: microbial recognition by immune receptors, signal transduction within plant cells, and immune execution directly suppressing pathogens. During the past three decades, many plant immune receptors and signaling components and their mode of action have been revealed, markedly advancing our understanding of the first two steps. Activation of immune signaling results in physical and chemical actions that actually stop pathogen infection. Nevertheless, this third step of plant immunity is under explored. In addition to immune execution by plants, recent evidence suggests that the plant microbiota, which is considered an additional layer of the plant immune system, also plays a critical role in direct pathogen suppression. In this review, we summarize the current understanding of how plant immunity as well as microbiota control pathogen growth and behavior and highlight outstanding questions that need to be answered.

A NAC-EXPANSIN module enhances maize kernel size by controlling nucellus elimination.

Maize early endosperm development is initiated in coordination with elimination of maternal nucellar tissues. However, the underlying mechanisms are largely unknown. Here, we characterize a major quantitative trait locus for maize kernel size and weight that encodes an EXPANSIN gene, ZmEXPB15. The encoded ß-expansin protein is expressed specifically in nucellus, and positively controls kernel size and weight by promoting nucellus elimination. We further show that two nucellus-enriched transcription factors (TFs), ZmNAC11 and ZmNAC29, activate ZmEXPB15 expression. Accordingly, these two TFs also promote kernel size and weight through nucellus elimination regulation, and genetic analyses support their interaction with ZmEXPB15. Importantly, hybrids derived from a ZmEXPB15 overexpression line have increased kernel weight, demonstrates its potential value in breeding. Together, we reveal a pathway modulating the cellular processes of maternal nucellus elimination and early endosperm development, and an approach to improve kernel weight.

Identification of a candidate gene underlying qHKW3, a QTL for hundred-kernel weight in maize.

KEY MESSAGE: qHKW3, a quantitative trait locus for hundred-kernel weight, harbors the proposed causal gene Zm00001d044081, encoding a homeobox-leucine zipper protein (ATHB-4) that might affect kernel size and weight. Kernel size and weight are key traits that contribute greatly to grain yield per year in maize (Zea mays). Here, we developed the chromosome segment substitution line (CSSL), H15-6-2, with smaller kernel size and lower kernel weight across environments compared to the background line Ye478. Histological analysis suggested that a slower kernel filling rate of H15-6-2 contributes to its small-kernel size and reduced hundred-kernel weight. We identified a quantitative trait locus (QTL) explaining 23% of the phenotypic variation in hundred-kernel weight. This QTL, qHKW3, was fine mapped to an interval of approximately 40.66-kb harboring the gene Zm00001d044081. The upstream sequence and its expression level of Zm00001d044081 in kernels at 6 days after pollination (DAP) showed obvious differences between the near-isogenic lines HKW3Ye478 and HKW3H15-6-2. We further confirmed the effects of the Zm00001d044081 promoter on maize kernel size and weight in an independent association mapping panel with 513 lines by candidate regional association analysis. We propose that Zm00001d044081, which encodes the homeobox-leucine zipper protein ATHB-4, is the causal gene of qHKW3, representing an attractive target for the genetic improvement of maize yield.

Knockdown NRPC2, 3, 8, NRPABC1 and NRPABC2 Affects RNAPIII Activity and Disrupts Seed Development in Arabidopsis.

RNA polymerase III (RNAPIII) contains 17 subunits forming 4 functional domains that control the different stages of RNAPIII transcription and are dedicated to the synthesis of small RNAs such as 5S rRNA and tRNAs. Here, we identified 23 genes encoding these subunits in Arabidopsis (Arabidopsis thaliana) and further analyzed 5 subunits (NRPC2, NRPC3, NRPC8, NRPABC1, and NRPABC2) encoded by 6 genes with different expression patterns and belonging to different sub-complexes. The knockdown of these genes repressed the expression of 5S rRNA and tRNAs, causing seed developmental arrest at different stages. Among these knockdown mutants, RNA-seq analysis revealed 821 common differentially expressed genes (DEGs), significantly enriched in response to stress, abscisic acid, cytokinins, and the jasmonic acid signaling pathway. Weighted gene co-expression network analysis (WGCNA) revealed several hub genes involved in embryo development, carbohydrate metabolic and lipid metabolic processes. We identified numerous unique DEGs between the mutants belonging to pathways, including cell proliferation, ribosome biogenesis, cell death, and tRNA metabolic processes. Thus, NRPC2, NRPC3, NRPC8, NRPABC1, and NRPABC2 control seed development in Arabidopsis by influencing RNAPIII activity and, thus, hormone signaling. Reduced expression of these subunit genes causes an insufficient accumulation of the total RNAPIII, leading to the phenotypes observed following the genetic knockdown of these subunits.

Fine mapping of a kernel length-related gene with potential value for maize breeding.

KEY MESSAGE: A key candidate gene for maize kernel length was fine mapped to an interval of 942 kb; the locus significantly increases kernel length (KL) and hundred-kernel weight (HKW). Kernel size is a major determinant of yield in cereals. Kernel length, one of the determining factors of kernel size, is a target trait for both domestication and artificial breeding. However, there are few reports of fine mapping and quantitative trait loci (QTLs)/cloned genes for kernel length in maize. In this project, a novel major QTL, named qKL9, controlling maize kernel length was identified. We verified the authenticity and stability of qKL9 via BC2F2 and BC3F1 populations, respectively, and ultimately mapped qKL9 to an ~ 942-kb genomic interval by testing the progenies of recombination events derived from BC3F2 and BC4F2 populations in multiple environments. Additionally, one new line (McqKL9-A) containing the ~ 942-kb segment was screened from the BC4F2 population. Combining transcriptome analysis between McqKL9-A and Mc at 6, 9 and 14 days after pollination and candidate regional association mapping, Zm00001d046723 was preliminarily identified as the key candidate gene for qKL9. Importantly, the replacement in the Mc line of the Mc's alleles by the V671's alleles in the qKL9 region improved the performances of single-cross hybrids obtained with elite lines, illustrating the potential value of this QTL for the genetic improvement in maize kernel-related traits. These findings facilitate molecular breeding for kernel size and cloning of the gene underlying qKL9, shedding light on the genetic basis of kernel size in maize.

ZmSRL5 is involved in drought tolerance by maintaining cuticular wax structure in maize.

Cuticular wax is a natural barrier on terrestrial plant organs, which protects plants from damages caused by a variety of stresses. Here, we report the identification and functional characterization of a cuticular-wax-related gene, Zea mays L. SEMI-ROLLED LEAF 5 (ZmSRL5). The loss-of-function mutant srl5, which was created by a 3,745 bp insertion in the first intron that led to the premature transcript, exhibited abnormal wax crystal morphology and distribution, which, in turn, caused the pleiotropic phenotypes including increased chlorophyll leaching and water loss rate, decreased leaf temperature, sensitivity to drought, as well as semi-rolled mature leaves. However, total wax amounts showed no significant difference between wild type and semi-rolled leaf5 (srl5) mutant. The phenotype of srl5 was confirmed through the generation of two allelic mutants using CRISPR/Cas9. ZmSRL5 encodes a CASPARIAN-STRIP-MEMBRANE-DOMAIN-LIKE (CASPL) protein located in plasma membrane, and highly expressed in developing leaves. Further analysis showed that the expressions of the most wax related genes were not affected or slightly altered in srl5. This study, thus, primarily uncovers that ZmSRL5 is required for the structure formation of the cuticular wax and could increase the drought tolerance by maintaining the proper cuticular wax structure in maize.

Loss of Function of an RNA Polymerase III Subunit Leads to Impaired Maize Kernel Development.

Kernel size is an important factor determining grain yield. Although a number of genes affecting kernel development in maize (Zea mays) have been identified by analyzing kernel mutants, most of the corresponding mutants cannot be used in maize breeding programs due to low germination or incomplete seed development. Here, we characterized small kernel7, a recessive small-kernel mutant with a mutation in the gene encoding the second-largest subunit of RNA polymerase III (RNAPΙΙΙ; NRPC2). A frame shift in ZmNRPC2 leads to a premature stop codon, resulting in significantly reduced levels of transfer RNAs and 5S ribosomal RNA, which are transcribed by RNAPΙΙΙ. Loss-of-function nrpc2 mutants created by CRISPR/CAS9 showed significantly reduced kernel size due to altered endosperm cell size and number. ZmNRPC2 affects RNAPIII activity and the expression of genes involved in cell proliferation and endoreduplication to control kernel development via physically interacting with RNAPIII subunits RPC53 and AC40, transcription factor class C1 and Floury3. Notably, unlike the semidominant negative mutant floury3, which has defects in starchy endosperm, small kernel7 only affects kernel size but not the composition of kernel storage proteins. Our findings provide novel insights into the molecular network underlying maize kernel size, which could facilitate the genetic improvement of maize in the future.

ZmSMK9, a pentatricopeptide repeat protein, is involved in the cis-splicing of nad5, kernel development and plant architecture in maize.

Maize kernel size and weight are essential contributors to its yield. So the identification of the genes controlling kernel size and weight can give us a chance to gain the yield. Here, we identified a small kernel mutant, Zea mays small kernel 9 (Zmsmk9), in maize. Cytological observation showed that the development of the endosperm and embryo was delayed in Zmsmk9 mutants at the early stages, resulting in a small kernel phenotype. Interestingly, despite substantial variation in kernel size, the germination of Zmsmk9 seeds was comparable to that of WT, and could develop into normal plants with upright leaf architecture. We cloned Zmsmk9 via map-based cloning. ZmSMK9 encodes a P-type pentatricopeptide repeat protein that targets to mitochondria, and is involved in RNA splicing in mitochondrial NADH dehydrogenase5 (nad5) intron-1 and intron-4. Consistent with the delayed development phenotype, transcriptome analysis of 12-DAP endosperm showed that starch and zeins biosynthesis related genes were dramatically down regulated in Zmsmk9, while cell cycle and cell growth related genes were dramatically increased. As a result, ZmSMK9 is a novel gene required for the splicing of nad5 intron-1 and intron-4, kernel development, and plant architecture in maize.